Methods and Compositions for Antibody Therapy

ABSTRACT

Methods and materials are provided for determining the responsiveness of a subject to a therapy, such as an antibody therapy and for selecting and/or for treating a subject based on an FcγRIIA polymorphism, or an FcγRIIIA polymorphism, or both an FcγRIIA polymorphism and an FcγRIIIA polymorphism, including where the treatment is an therapy, such as rituximab. Methods and materials are also provided for designing, making, screening, testing and/or administering antibodies as well as for optimizing antibody therapies based upon a subject&#39;s FcγRIIA polymorphism, or FcγRIIIA polymorphism, or both the FcγRIIA polymorphism and the FcγRIIIA polymorphism.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/298,661, filed Oct. 27, 2008, which is a national stage applicationunder 35 U.S.C. §371 of International application PCT/US2007/067663,filed Apr. 27, 2007, which claims benefit under 35 U.S.C. §119(e) ofU.S. provisional patent application No. 60/795,957, filed Apr. 27, 2006.The entire contents of each cited application is incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING

The official copy of the sequence listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “PMI-001US_ST25.txt”, a creation date of Oct. 28, 2013, anda size of 59.4 kilobytes. The sequence listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND

A number of antibodies have been developed, including for their use intherapies for a variety of diseases, disorders or conditions. Forexample, in the fall of 1997, the anti-CD20 monoclonal antibody,rituximab (currently sold under the brand name RITUXAN®), was approvedfor the treatment of refractory or relapsed low-grade B-cellnon-Hodgkin's lymphoma (NHL). Rituximab has since become a mainstay oftreatment for low-grade NHL and over 400,000 patients worldwide havebeen treated with rituximab. Despite this extensive clinical experience,the mechanism of action of rituximab remains unclear, as does the natureof resistance.

Rituximab is a chimeric antibody consisting of a murine CD20-bindingvariable region linked to human IgG₁ constant region. CD20 is a cellsurface protein expressed on B-lymphocytes. CD20 has four transmembranedomains and has been proposed to act as an ion channel; however, thefunction of CD20 remains poorly understood. Phase II trials of rituximabin people with refractory or relapsed low grade or follicular NHLdemonstrated a 50% response rate. While the nature of de novo resistanceto rituximab is unclear, such resistance is very rarely due to loss ofthe CD20 antigen, which cannot be shed or internalized and is rarelydown-regulated. Despite these properties of CD20, acquired resistance torituximab is common in that only half of patients previously respondingto rituximab will respond to a second course of treatment.

An effective and practical diagnostic protocol which could provideinformation as to whether a patient is or is not responsive to atherapy, including an antibody therapy such as rituximab therapy, wouldbe desirable for a number of reasons, including avoidance of delays inalternative treatments, elimination of exposure to adverse effects ofthe therapy and reduction of unnecessary expense. As such, there isinterest in the development of protocols that can accurately predictwhether or not a patient is responsive to such therapies. There is alsoan interest in the development of antibodies and antibody therapies thatwould be effective or more effective in patients that werenon-responsive or poorly responsive to a particular therapy.

SUMMARY

Methods and materials are provided for determining the responsiveness ofa subject to a therapy, such as an antibody therapy and for selectingand/or for treating a subject based on an FcγRIIA polymorphism, or anFcγRIIIA polymorphism, or both an FcγRIIA polymorphism and an FcγRIIIApolymorphism, including where the treatment is an therapy, such asrituximab. Methods and materials are also provided for designing,making, screening, testing and/or administering antibodies as well asfor optimizing antibody therapies based upon a subject's FcγRIIApolymorphism, or FcγRIIIA polymorphism, or both the FcγRIIA polymorphismand the FcγRIIIA polymorphism.

Methods and compositions are provided for determining whether a subjectsuffering from a neoplastic condition is responsive to an antineoplastictherapy, such as antibody therapy, e.g., rituximab therapy. Inpracticing the subject methods, the subject is genotyped to determinewhether the subject has at least one favorable FcγR polymorphism, e.g.,the H/H¹³¹ genotype in FcγRIIA or the V/V¹⁵⁸ genotype in FcγRIIIA. Inaddition, reagents, devices and kits thereof, that find use inpracticing the subject methods are provided.

Methods are provided for determining the degree of responsiveness that asubject having an ADCC-treatable disease or disorder will have to anantibody therapy for the disease or disorder by genotyping the subjectfor an FcγRIIA polymorphism to obtain a first result, genotyping thesubject for an FcγRIIIA polymorphism to obtain a second result,assigning the subject to one of more than three categories of treatmentresponse and/or employing the first and second results to determine thedegree of the responsiveness of the subject to the antibody therapy.

Methods are provided for selecting a specific Fc variant antibodytherapy from a set of two or more Fc variant antibody therapies for usein treating subjects having an ADCC-treatable disease by genotyping thesubjects for an FcγRIIA polymorphism to classify patient population intothree groups (e.g., H/H¹³¹, H/R¹³¹, R/R¹³¹), genotyping the subjects foran FcγRIIIA polymorphism to classify patient population into threegroups (e.g., V/V¹⁵⁸, V/F¹⁵⁸, F/F¹⁵⁸), classifying the subjects intonine patient groups I-IX based on the first and second results, andemploying the first and second results to select a specific Fc variantantibody therapy for the patient group such that the degree of treatmentresponse to antibody therapy in the patient group is improved. Subjectsmay be classified into nine groups based on their genotype, including:V/V¹⁵⁸, H/H¹³¹ (Group-I); V/F¹⁵⁸, H/H¹³¹ (Group-II); F/F¹⁵⁸, H/H¹³¹(Group-III); V/V¹⁵⁸, H/R¹³¹ (Group-IV); V/F¹⁵⁸, H/R¹³¹ (Group-V);F/F¹⁵⁸, H/R¹³¹ (Group-VI); V/V¹⁵⁸ R/R¹³¹ (Group-VII); V/F¹⁵⁸, R/R¹³¹(Group-VIII); and F/F¹⁵⁸, R/R¹³¹ (Group-IX).

Methods are also provided for making a set of related antibodies bymodifying the amino acid sequence of at least one Fc region amino acidresidue in a parent antibody, such that the modified Fc region exhibitsenhanced binding affinity to at least one Fc receptor encoded by an Fcreceptor gene of a first genotype, compared to the Fc binding affinityof the parent antibody, to generate a first Fc variant antibody; and/ormodifying at least one Fc region amino acid residue in a parentantibody, such that the modified constant region exhibits enhancedbinding affinity to at least one Fc receptor encoded by an Fc receptorgene of a second genotype, compared to the Fc binding affinity of theparent antibody, to generate a second Fc variant antibody, wherein thefirst and second Fc variant antibodies have the same antigenspecificity.

Also provided are kits for use in determining responsiveness to anantibody therapy in a patient which include an element for genotyping asample to identify an FcγRIIA polymorphism, an element for genotypingthe sample to identify an FcγRIIIA polymorphism, or an element forgenotyping the sample to identify an FcγRIIIA polymorphism and anFcγRIIA, and a reference that correlates a genotype with predictedtherapeutic response to a therapeutic antibody.

Methods are provided for of treating an ADCC-treatable disease ordisorder in an individual by determining a category of therapeuticresponsiveness to an antibody therapy by genotyping the individual foran FcγRIIA polymorphism and an FcγRIIIA polymorphism, wherein theFcγRIIA polymorphism and the FcγRIIIA polymorphism together indicate adegree of therapeutic responsiveness; selecting an antibody from a setof related antibodies, wherein members of the set of related antibodieshave the same antigen binding specificity, and wherein the members ofthe set of related antibodies differ in binding affinity to an FcγRIIAand/or an FcγRIIIA and/or differ in in vitro ADCC function; andadministering an effective amount of the antibody to the individual.

Methods are provided for determining the degree of responsiveness to anantibody-dependent cell-mediated cytotoxicity antibody therapy bygenotyping the subject for two or more Fcγ receptor polymorphisms andemploying the first and second Fcγ receptor polymorphisms to determinethe degree of the responsiveness of the subject to the antibody therapy.

Methods are also provided for generating a set of Fc variant antibodiesby amplifying a nucleic acid comprising a nucleotide sequence encodingan Fc region of an antibody, wherein the amplifying is carried out witha set of primers that encode all nineteen amino acid variants at asingle residue of the Fc region, to generate a set of variant nucleicacids encoding nineteen amino acid substitution variants at the singleresidue of the Fc region; transcribing and translating each of thevariant nucleic acids in vitro, to generate a set of Fc variants; and/orc) selecting from the set an Fc variant having altered FcR bindingactivity compared to a reference Fc, generating a set of selected Fcvariants.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA H/H¹³¹genotype; or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA H/H¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA H/H¹³¹genotype; or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA H/H¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, or an FcγRIIAH/H¹³¹ genotype; or both an FcγRIIIA F/F¹⁵⁸ genotype and an FcγRIIAH/H¹³¹ genotype and (b) administering the antibody to the patientselected in step (a).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype; or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are also provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype, or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype, or both an FcγRIIIA F/F¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA R/R¹³¹genotype, or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA R/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are also provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA R/R¹³¹genotype, or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA R/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

Methods are provided for treating a patient with an antibody,comprising: (a) selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, anFcγRIIA R/R¹³¹ genotype, or both an FcγRIIIA F/F¹⁵⁸ genotype and anFcγRIIA R/R¹³¹ genotype and (b) administering the antibody to thepatient selected in step (a).

Methods are also provided for classifying a subject having anADCC-treatable disease or disorder into one of more than threecategories of responsiveness to an antibody therapy by genotypingsubjects for an FcγRIIA polymorphism and an FcγRIIIA polymorphism,wherein the subjects have or had the ADCC-treatable disease or disorderand are or were administered antibody therapy for the disease ordisorder; classifying each subject based on its FcγRIIA polymorphism andFcγRIIIA polymorphism to one of three or more categories ofresponsiveness to the antibody therapy; genotyping the subject for anFcγRIIA polymorphism and an FcγRIIIA polymorphism; identifying agenotype from (a) that is identical to the genotype from the subject instep (c), wherein the subject is classified into a category ofresponsiveness to the antibody therapy for the disease or disordercorresponding with a subject having an identical FcγRIIA polymorphismand an identical FcγRIIIA polymorphism.

Methods are provided for determining the degree of responsiveness that asubject having an ADCC-treatable disease or disorder will have to anantibody therapy for the disease or disorder by genotyping the subjectfor an FcγRIIA polymorphism and an FcγRIIIA polymorphism; andidentifying a genotype associated with a particular degree ofresponsiveness to the antibody therapy from a reference that isidentical to the genotype from the test subject, wherein the testsubject is determined to have a degree of responsiveness to the antibodytherapy for the disease or disorder corresponding to the level ofresponsiveness associated with the reference having an identical FcγRIIApolymorphism and an identical FcγRIIIA polymorphism.

Methods are also provided for determining the degree of responsivenessthat a test subject having an ADCC-treatable disease or disorder willhave to an antibody therapy for the disease or disorder by (a)genotyping subjects for an FcγRIIA polymorphism and an FcγRIIIApolymorphism, wherein the subjects have or had the ADCC-treatabledisease or disorder and are or were administered antibody therapy forthe disease or disorder; (b) classifying each subject based on itsFcγRIIA polymorphism and FcγRIIIA polymorphism to one of more than threecategories of responsiveness to the antibody therapy; (c) genotyping thetest subject for an FcγRIIA polymorphism and an FcγRIIIA polymorphism;and (d) identifying a genotype from (a) that is identical to thegenotype from the test subject in step (c), wherein the test subject isdetermined to have a degree of responsiveness to the antibody therapyfor the disease or disorder corresponding to the level of responsivenessassociated with a subject having an identical FcγRIIA polymorphism andan identical FcγRIIIA polymorphism.

Also provided are kits for use in determining responsiveness to anantibody therapy in a patient which include an element for genotypingthe sample to identify an FcγRIIA polymorphism; an element forgenotyping the sample to identify an FcγRIIIA polymorphism; and areference that correlates a genotype in the patient with one of morethan three predicted therapeutic responses to the antibody therapy.

Methods are provided for selecting a specific variant antibody therapyfrom a set of two or more variant antibody therapies for use intreatment of subjects having an ADCC-treatable disease by genotyping thesubjects for an FcγRIIA polymorphism and an FcγRIIIA polymorphism,classifying the subjects into one of more than three categories ofresponsiveness based on their FcγRIIA polymorphism and their FcγRIIIApolymorphism, and selecting a specific variant antibody therapy for thesubjects such that the degree of responsiveness to the antibody therapyin the subjects is improved from the degree of responsiveness obtainedwith another variant antibody.

Methods are also provided for treating an ADCC-treatable disease ordisorder in a subject by genotyping the subject for an FcγRIIApolymorphism and an FcγRIIIA polymorphism, classifying the subject intoone of more than three categories of therapeutic responsiveness to anantibody therapy based on the FcγRIIA polymorphism and the FcγRIIIApolymorphism, selecting an antibody with a preferred degree oftherapeutic responsiveness from a set of related antibodies, whereinmembers of the set of related antibodies have the same antigen bindingspecificity, and wherein the members of the set of related antibodiesdiffer in binding affinity to an FcγRIIA and/or an FcγRIIIA and/ordiffer in in vitro ADCC function, and administering a therapeuticallyeffective amount of the antibody to the subject, wherein, the antibodytreats the ADCC-treatable disease or disorder in the subject.

Methods are provided for making a set of related antibodies capable ofmodulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorderby modifying the amino acid sequence of at least one amino acid residuein a parent antibody, such that the modified parent antibody exhibitsenhanced binding affinity to at least one Fc receptor encoded by an Fcreceptor gene of a first genotype, compared to the Fc binding affinityof the parent antibody, to generate a first variant antibody; andmodifying at least one amino acid residue in a parent antibody, suchthat the modified parent antibody exhibits enhanced binding affinity toat least one Fc receptor encoded by an Fc receptor gene of a secondgenotype, compared to the Fc binding affinity of the parent antibody, togenerate a second variant antibody, wherein the first and second variantantibodies have the same antigen specificity and are capable ofmodulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorder.

Methods are provided for generating a set of variant antibodies capableof modulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorderby amplifying a nucleic acid comprising a nucleotide sequence encoding aregion of an antibody, wherein the amplifying is carried out with a setof primers that encode all nineteen amino acid variants at a singleresidue of the region, to generate a set of variant nucleic acidsencoding nineteen amino acid substitution variants at the single residueof the region, transcribing and translating each of the variant nucleicacids in vitro, to generate a set of variants, and/or selecting from theset an variant having altered FcR binding activity compared to areference region, generating a set of selected variants, wherein thefirst and second variant antibodies have the same antigen specificityand are capable of modulating the responsiveness of a subject having anADCC-treatable disease or disorder to an antibody therapy for thedisease or disorder. In some embodiments, the method includesdetermining in vitro ADCC activity of the selected variant.

Methods are also provided for modulating the responsiveness of a subjecthaving an ADCC-treatable disease or disorder to an antibody therapy forthe disease or disorder by genotyping the subject for an FcγRIIApolymorphism and an FcγRIIIA polymorphism, classifying the subject intoone of more than three categories of therapeutic responsiveness to anantibody therapy based on the FcγRIIA polymorphism and the FcγRIIIApolymorphism, selecting an antibody from a set of related antibodies,wherein members of the set of related antibodies have the same antigenbinding specificity, and wherein the members of the set of relatedantibodies differ in binding affinity to an FcγRIIA and/or an FcγRIIIAand/or differ in in vitro ADCC function, and administering atherapeutically effective amount of the antibody to the subject, whereinthe antibody modulates the responsiveness of the subject having anADCC-treatable disease or disorder to an antibody therapy for thedisease or disorder.

Methods are provided for enhancing antibody dependent cell mediatedcytotoxicity (ADCC) activity of an antibody for use in treatment of asubject having an ADCC-treatable disease by genotyping the subject foran FcγRIIA polymorphism and an FcγRIIIA polymorphism, selecting an Fcnucleotide sequence for the antibody that has optimal ADCC for theFcγRIIA polymorphism and FcγRIIIA polymorphism, and modifying theantibody to include the optimal Fc sequence for the subject's genotype,wherein the ADCC activity of the antibody is enhanced by using theoptimal Fc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Rituximab-induced antibody-dependent cell-mediated cytotoxicity(ADCC). The scatter plot in the left column of each Group represents thedegree of rituximab-induced ADCC (effector/target ratio at 30:1) ofindividual tumors. The bars represent the mean and standard deviationsin each Group. NR, nonresponder; PR, partial responder; CR, completeresponder or complete response unconfirmed.

FIG. 2: Kaplan-Meier estimates of progression free survival by IgG Fcreceptor IIIA (FcγRIIIA) 158 valine (V)/phenylalanine (F) polymorphism.Progression-free survival (PFS) curves were plotted by FcγRIIIA V/F¹⁵⁸genotype on all 87 patients. F carriers represent patients with eitherV/F¹⁵⁸ or F/F¹⁵⁸ genotype. TTP, median time to progression.

FIG. 3: Kaplan-Meier estimates of progression-free survival (PFS) by IgGFc receptor HA (FcγRIIA) 131 histidine (H)/arginine (R) polymorphism.PFS curves were plotted by FcγRIIA H/R¹³¹ genotype on all 87 patients. Rcarriers represent patients with either H/R¹³¹ or R/R¹³¹ genotype. TTP,median time to progression.

FIG. 4: Progression free survival (PFS) by IgG Fc receptor IIIa(FcγRIIIA) V/F¹⁵⁸ and FcγRIIA H/R¹³¹ polymorphisms. PFS curves wereplotted by FcγRIIIA V/F¹⁵⁸ and FcγRIIA H/R¹³¹ genotype. Others representpatients without either FcγRIIIA V/V¹⁵⁸ or FcγRIIA H/H¹³¹ genotype. TTP,median time to progression.

FIGS. 5, 6, 7, 8, 9, 10 and 11 provide Tables 1 to 7 referred to in theExperimental Section, below.

FIGS. 12A-D: Amino acid sequences of Fc receptors and IgGs. FIG. 12Adepicts an amino acid sequence alignment of FcγRIIIA (SEQ ID NO:26) andFcγRIIA (SEQ ID NO:27) from residues 83-170. Identical residues betweenthe receptors are aligned, and the FcR residues that contact Fcl are inbold. According to the numbering system used in crystal structurestudies, the Valine at position 155 of FcγRIIIA is the residue referredto herein as V¹⁵⁸. The residues H/R¹³¹ and V/I¹⁵⁸ are underlined. FIG.12B depicts an amino acid sequence of hIgG1 from residues 229-444 (SEQID NO:34). Key binding motifs in the Fc region are in bold. FIG. 12Cdepicts a structure-based sequence alignment of FcγRIII (FcγR1—SEQ IDNO:36, SEQ ID NO: 37, SEQ ID NO:38; FcγRIIa-HR—SEQ ID NO: 39, SEQ ID NO:40, and SEQ ID NO: 41; FcγRIIa-LR—SEQ ID NO: 42, SEQ ID NO: 43, and SEQID NO: 44; FcγRIIb—SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;FcγRIa—SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50; FcγRIII-V—SEQ IDNO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; and FcγRIII-F—SEQ ID NO: 54,SEQ ID NO: 55, and SEQ ID NO: 56) and hIgG1 (IgG₁—SEQ ID NO: 57, SEQ IDNO: 58, and SEQ ID NO: 59; IgG₂—SEQ ID NO: 60, SEQ ID NO: 61, and SEQ IDNO: 62; IgG₃—SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65; IgG₄—SEQID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68; IgE—SEQ ID NO: 69, SEQ IDNO: 70, and SEQ ID NO: 71) with their respective homologues. HRindicates high responders; LR indicates low responders. FcγRIIIA-V, V¹⁵⁸allele; FcγRIIIA-F, F¹⁵⁸ allele. FIG. 12D depicts Fc Walking: Thisinvolves bi-directional scanning saturation mutagenesis of approximately5-10 residues, one residue at a time, on both sides of the “binding”motifs of the hFc regions namely lower hinge region (SEQ ID NO:30), B/Cloop (SEQ ID NO:31), C/E loop (SEQ ID NO:32), and the F/G loop (SEQ IDNO:33).

FIG. 13: Table depicting an analysis of FcγRIIIA and FcγRIIApolymorphisms in B-NHL patients.

FIG. 14: Table depicting prevalence of FcγRIIIA and FcγRIIApolymorphisms in B-NHL patients (Weng), healthy U.S. Caucasians(Lehrnbecher), healthy U.S. African Americans (Lehrnbecher) and healthyNorwegians (Torkildsen).

FIG. 15: Alignment of Antibody Fc Regions: Table comparing thenucleotide sequence of the Fc regions of Rituxan® (SEQ ID NO:1),Remicade® (SEQ ID NO:12), Erbitux® (SEQ ID NO:13), Campath® (SEQ IDNO:14) and Herceptin® (SEQ ID NO:15) (Prepared with CLUSTAL W (1.83);Mismatches are indicated by the absence of a “*” underneath thealignment.

FIG. 16: SSM in rituximab V_(L) CDR2 region (SEQ ID NO:18).

FIG. 17: Simultaneous SSM of the CDR regions of V_(L) (SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 19) and V_(H) (SEQ ID NO: 17, SEQ ID NO: 35,SEQ ID NO: 20) sequences of Rituximab. Optimal mutations identified inthe CDR regions are highlighted (V_(L) CDR1—SEQ ID NO:21; V_(L) CDR2—SEQID NO:22; V_(H) CDR3—SEQ ID NO:23).

FIG. 18: Sequence Comparison of the Hinge Region of human IgG₃ and humanIgG₁. Numbers correspond to those of IgG₁ Eu-residues 215 to 254(Edelman et. al., Proc. Natl. Acad. Sci. USA 63:78, 1969). The IgG₃hinge region (SEQ ID NO:24) is about 4 times larger than the counterpartregion of IgG₁ (SEQ ID NO:25), IgG₂, and IgG₄. The insertion sequence ofthe IgG₃ hinge region consists of an N-terminal 17-residue segmentfollowed by a 15-residue segment that is identically and consecutivelyrepeated three times (Michaelsen et. al., J. Biol. Chem. 252:883, 1977).

DEFINITIONS

A polynucleotide has a certain percent “sequence identity” to anotherpolynucleotide, meaning that, when aligned, that percentage of bases arethe same, and in the same relative position, when comparing the twosequences. Sequence similarity can be determined in a number ofdifferent manners. To determine sequence identity, sequences can bealigned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g.,Altschul et al., 1990, J. Mol. Biol. 215:403-10. Another alignmentalgorithm is FASTA, available in the Genetics Computing Group (GCG)package, from Madison, Wis., USA, a wholly owned subsidiary of OxfordMolecular Group, Inc. Other techniques for alignment are described inMethods in Enzymology, vol. 266: Computer Methods for MacromolecularSequence Analysis (1996), ed. Doolittle, Academic Press, Inc., adivision of Harcourt Brace & Co., San Diego, Calif., USA. Of particularinterest are alignment programs that permit gaps in the sequence. TheSmith-Waterman is one type of algorithm that permits gaps in sequencealignments. See, e.g., Meth. Mol. Biol. 70: 173-187 (1997). Also, theGAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See, e.g., J. Mol. Biol. 48: 443-453(1970).

A nucleic acid is “hybridizable” to another nucleic acid, such as acDNA, genomic DNA, or RNA, when a single stranded form of the nucleicacid can anneal to the other nucleic acid under the appropriateconditions of temperature and solution ionic strength. Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J.and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.

Hybridization conditions and post-hybridization washes are useful toobtain the desired determine stringency conditions of the hybridization.One set of illustrative post-hybridization washes is a series of washesstarting with 6×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer),0.5% SDS at room temperature for 15 minutes, then repeated with 2×SSC,0.5% SDS at 45° C. for 30 minutes, and then repeated twice with 0.2×SSC,0.5% SDS at 50° C. for 30 minutes. Other stringent conditions areobtained by using higher temperatures in which the washes are identicalto those above except for the temperature of the final two 30 minutewashes in 0.2×SSC, 0.5% SDS, which is increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C. Another example of stringent hybridization conditions ishybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5mM sodium citrate). Another example of stringent hybridizationconditions is overnight incubation at 42° C. in a solution: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C. Stringent hybridization conditionsand post-hybridization wash conditions are hybridization conditions andpost-hybridization wash conditions that are at least as stringent as theabove representative conditions.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of the melting temperature (Tm) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher Tm) of nucleic acid hybridizations decreases inthe following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greaterthan 100 nucleotides in length, equations for calculating Tm have beenderived (See, e.g., Sambrook et al., supra, 9.50-9.51). Forhybridizations with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (See, e.g., Sambrook et al.,supra, 11.7-11.8). In some embodiments, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Illustrative minimumlengths for a hybridizable nucleic acid are: at least about 15nucleotides; at least about 20 nucleotides; and at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides or polypeptides. The term “biological sample”encompasses a clinical sample, and also includes cells in culture, cellsupernatants, cell lysates, serum, plasma, biological fluid, and tissuesamples.

As used herein, the term “target nucleic acid region” or “target nucleicacid” or “target molecules” refers to a nucleic acid molecule with a“target sequence” to be detected (e.g., by amplification). The targetnucleic acid may be either single-stranded or double-stranded and may ormay not include other sequences besides the target sequence (e.g., thetarget nucleic acid may or may not include nucleic acid sequencesupstream or 5′ flanking sequence, may or may not include downstream or3′ flanking sequence, and in some embodiments may not include eitherupstream (5′) or downstream (3′) nucleic acid sequence relative to thetarget sequence. Where detection is by amplification, these othersequences in addition to the target sequence may or may not be amplifiedwith the target sequence.

The term “target sequence” or “target nucleic acid sequence” refers tothe particular nucleotide sequence of the target nucleic acid to bedetected (e.g., through hybridization and/or amplification). The targetsequence may include a probe-hybridizing region contained within thetarget molecule with which a probe will form a stable hybrid underdesired conditions. The “target sequence” may also include thecomplexing sequences to which the oligonucleotide primers complex and beextended using the target sequence as a template. Where the targetnucleic acid is originally single-stranded, the term “target sequence”also refers to the sequence complementary to the “target sequence” aspresent in the target nucleic acid. If the “target nucleic acid” isoriginally double-stranded, the term “target sequence” refers to boththe plus (+) and minus (−) strands. Moreover, where sequences of a“target sequence” are provided herein, it is understood that thesequence may be either DNA or RNA. Thus where a DNA sequence isprovided, the RNA sequence is also contemplated and is readily providedby substituting “T” of the DNA sequence with “U” to provide the RNAsequence.

The term “primer” or “oligonucleotide primer” as used herein, refers toan oligonucleotide which acts to initiate synthesis of a complementarynucleic acid strand when placed under conditions in which synthesis of aprimer extension product is induced, e.g., in the presence ofnucleotides and a polymerization-inducing agent such as a DNA or RNApolymerase and at suitable temperature, pH, metal concentration, andsalt concentration. Primers are in some embodiments of a lengthcompatible with their use in synthesis of primer extension products, andare in some embodiments are in the range of between 8 nucleotides to 100nucleotides in length, such as 10 to 75 nucleotides, 15 to 60nucleotides, 15 to 40 nucleotides, 18 to 30 nucleotides, 20 to 40nucleotides, 21 to 50 nucleotides, 22 to 45 nucleotides, or 25 to 40nucleotides, and so on. In some embodiments, a primer has a length inthe range of between 18-40 nucleotides, 20-35 nucleotides, or 21-30nucleotides, and any length between the stated ranges. In someembodiments, primers are in the range of between 10-50 nucleotides long,such as 15-45 nucleotides long, 18-40 nucleotides long, 20-30nucleotides long, 21-25 nucleotides long and so on, and any lengthbetween the stated ranges. In some embodiments, the primers are not morethan about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

Primers are in some embodiments single-stranded for maximum efficiencyin amplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is usually first treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step can be effected by heat, but may alternatively becarried out using alkali, followed by neutralization. Thus, a “primer”is complementary to a template, and complexes by hydrogen bonding orhybridization with the template to give a primer/template complex forinitiation of synthesis by a polymerase, which is extended by theaddition of covalently bonded bases linked at its 3′ end complementaryto the template in the process of DNA synthesis.

A “primer pair” as used herein refers to first and second primers havingnucleic acid sequence suitable for nucleic acid-based amplification of atarget nucleic acid. Such primer pairs generally include a first primerhaving a sequence that is the same or similar to that of a first portionof a target nucleic acid, and a second primer having a sequence that iscomplementary to a second portion of a target nucleic acid to providefor amplification of the target nucleic acid or a fragment thereof.Reference to “first” and “second” primers herein is arbitrary, unlessspecifically indicated otherwise. For example, the first primer can bedesigned as a “forward primer” (which initiates nucleic acid synthesisfrom a 5′ end of the target nucleic acid) or as a “reverse primer”(which initiates nucleic acid synthesis from a 5′ end of the extensionproduct produced from synthesis initiated from the forward primer).Likewise, the second primer can be designed as a forward primer or areverse primer.

As used herein, the term “probe” or “oligonucleotide probe”, usedinterchangeable herein, refers to a structure comprised of apolynucleotide, as defined above, which contains a nucleic acid sequencecomplementary to a nucleic acid sequence present in the target nucleicacid analyte (e.g., a nucleic acid amplification product). Thepolynucleotide regions of probes may be composed of DNA, and/or RNA,and/or synthetic nucleotide analogs. Probes are in some embodiments of alength compatible with its use in specific detection of all or a portionof a target sequence of a target nucleic acid, and are in someembodiments in the range of between 8 nucleotides to 100 nucleotides inlength, such as 8 to 75 nucleotides, 10 to 74 nucleotides, 12 to 72nucleotides, 15 to 60 nucleotides, 15 to 40 nucleotides, 18 to 30nucleotides, 20 to 40 nucleotides, 21 to 50 nucleotides, 22 to 45nucleotides, or 25 to 40 nucleotides, and so on. In some embodiments, aprobe has a length in the range of between 18-40 nucleotides, 20-35nucleotides, or 21-30 nucleotides long, and any length between thestated ranges. In some embodiments, a probe is in the range of between10-50 nucleotides long, such as 15-45 nucleotides, 18-40 nucleotides,20-30 nucleotides, 21-28 nucleotides, or 22-25 nucleotides, and so on,and any length between the stated ranges. In some embodiments, theprimers are not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

Probes contemplated herein include probes that include a detectablelabel. As used herein, the terms “label” and “detectable label” refer toa molecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes,metal ions, metal sols, ligands (e.g., biotin, avidin, streptavidin orhaptens) and the like. The term “fluorescer” refers to a substance or aportion thereof which is capable of exhibiting fluorescence in thedetectable range.

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer “hybridizes” with target (template), such complexes (or hybrids)are sufficiently stable to serve the priming function required by, e.g.,the DNA polymerase to initiate DNA synthesis.

The term “stringent conditions” refers to conditions under which aprimer will hybridize preferentially to, or specifically bind to, itscomplementary binding partner, and to a lesser extent to, or not at allto, other sequences. Put another way, the term “stringent hybridizationconditions” as used herein refers to conditions that are compatible toproduce duplexes on an array surface between complementary bindingmembers, e.g., between probes and complementary targets in a sample,e.g., duplexes of nucleic acid probes, such as DNA probes, and theircorresponding nucleic acid targets that are present in the sample, e.g.,their corresponding mRNA analytes present in the sample.

Exemplary stringent conditions typically will be those in which the saltconcentration is at least about 0.01 to 1.0 M sodium ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes (e.g., 10 to 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. Thus, in a preferred embodiment, thehybridized array may be washed at successively higher stringencysolutions and read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above that the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular oligonucleotide probes of interest.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining,”“measuring,” “evaluating,” “assessing,” and “assaying” are usedinterchangeably and includes quantitative and qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present,and/or determining whether it is present or absent. As used herein, theterms “determining,” “measuring,” and “assessing,” and “assaying” areused interchangeably and include both quantitative and qualitativedeterminations.

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” is aterm well understood in the art, and refers to a cell-mediated reactionin which non-specific cytotoxic cells that express Fc receptors (FcRs)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. Non-specific cytotoxic cells that mediate ADCCinclude natural killer (NK) cells, macrophages, monocytes, neutrophils,and eosinophils.

As used herein, the term “ADCC-dependent antibody therapy” refers to atherapy involving use of antibody that comprises an antigen-bindingdomain and an Fc region that binds an FcR of a cytotoxic effector cell,where binding of the antibody to a target cell results in killing of thetarget cell via ADCC, and where killing of the target cell(s) providesfor a therapeutic effect in an individual.

An “ADCC-treatable disease, condition, or disorder,” as used herein, isa disease, condition, or disorder that is treated with a therapeuticantibody that mediates ADCC, thereby treating the disease, condition, ordisorder. ADCC-treatable diseases, conditions, and disorders include,but are not limited to, a neoplastic disease; an autoimmune disease; amicrobial infection; and allograft rejection.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe γ chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FcR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (Ravetch J. V. et al., 2001, Annu. Rev.Immunol. 19:275-90). FcγRs, designated FcγRI (CD64), FcγRII (CD32), andFcγRIII (CD16), are encoded by distinct genes although they shareextensive sequence homology.

FcγRII (CD32) is a 40 KDa integral membrane glycoprotein which bindscomplexed IgG; and exhibits only low affinity for monomeric Ig (appr.10⁶ M⁻¹).

FcγRII is the most widely expressed FcγR, present on all hematopoieticcells, including monocytes, macrophages, B cells, NK cells, neutrophils,mast cells, and platelets (Cohen-Solal et al., 2004, Immunol. Lett.92:199-205). FcγRII has only two immunoglobulin-like regions in itsimmunoglobulin binding chain and hence a much lower affinity for IgGthan FcγRI. There are three human FcγRII genes (FcγRIIA, FcγRIIB,FcγRIIC), all of which bind IgG in aggregates or immune complexes.Distinct differences within the cytoplasmic domains of FcγRIIA andFcγRIIB create two functionally heterogeneous responses to receptorligation. FcγRIIA initiates intracellular signaling leading to cellactivation such as phagocytosis and respiratory burst, whereas FcγRIIBinitiates inhibitory signals leading to the inhibition of B-cellactivation.

FcγRIII (CD16) is a Type-I membrane protein that exists as two isoforms:FcγRIIIA and FcγRIIIB. Both FcγRIIIA and FcγRIIIB are low affinityreceptors. FcγRIIIA, an activating receptor, is expressed on NK cells,macrophages, monocytes, and dendritic cells; FcγRIIIB, an inhibitoryform, is expressed on neutrophils. All FcγRs bind the same region on IgGFc, yet with differing high (FcγRI) and low (FcγRII and FcγRIII)affinities (Sondermann et al., 2001, J. Mol. Biol. 309:737-749).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacological and/or physiological effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) increasing survival time; (b) decreasing the risk of deathdue to the disease; (c) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (d) inhibiting the disease, i.e., arresting itsdevelopment (e.g., reducing the rate of disease progression); and (e)relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient,” usedinterchangeably herein, refer to a mammal, including primates, rodents,livestock, pets, horses, etc. In some embodiments, an individual is ahuman.

A “functional Fc region” possesses an “effector function” of a native Fcregion, e.g., ADCC activity. Although the boundaries of the Fc region ofan immunoglobulin heavy chain may vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus. The Fc regionof an immunoglobulin generally comprises two constant domains, CH2 andCH3. A “native Fc region sequence” comprises an amino acid sequenceidentical to the amino acid sequence of an Fc region found in nature.Native human Fc region sequences include, but are not limited to, thehuman IgG1 Fc region (non-A and A allotypes); human IgG2 Fc region;human IgG3 Fc region; and human IgG4 Fc region as well as naturallyoccurring variants thereof.

The term “Fc Walking” as used herein refers to an antibody engineeringprocedure by which the amino acid residues in the Fc region areselectively mutated around one or more of the lower hinge region, B/Cloop, C′/E loop, and the F/G loop. Fc Walking involves bi-directionalmutagenesis of approximately 5-10 residues, one residue at a time, onboth sides of the Fc-FcR “binding” motifs with an objective of enhancingthe Fc-FcR binding affinity and the ADCC activity of IgG variants. As anexample, Fc Walking would cover the sequence stretch, L²³⁴-S²³⁹, as wellas the residues upstream (C²²⁹-E²³³) and downstream (V²⁴⁰-P²⁴⁵) of thisstretch. One such antibody engineering procedure that can be employedfor Fc Walking is in vitro scanning saturation mutagenesis.

The term “Fc variant antibody” refers to an antibody that differs inamino acid sequence by at least one amino acid, compared to a referenceantibody (where a reference antibody is also referred to as a “parentantibody”). In some embodiments, the Fc variant antibody is a monoclonalantibody (MAb); in these embodiments, the Fc variant antibody isreferred to as an “Fc variant MAb.” An Fc variant antibody may havealtered FcR binding properties (e.g., enhanced FcR binding affinity),and/or altered ADCC activity, and/or altered effector function.

The term “enhanced affinity” is used to denote the significant increasein binding of the Fc variant antibody to one or more FcRs, compared tothe binding affinity of the parent antibody for the same FcR(s). Anincrease of 10% or more in binding affinity over the parent antibody isconsidered significant.

The terms “cancer,” “neoplasm,” “hyperproliferative cell,” and “tumor”are used interchangeably herein to refer to cells which exhibitrelatively autonomous growth, so that they exhibit an aberrant growthphenotype characterized by a significant loss of control of cellproliferation. Cancerous cells can be benign or malignant. Viralinfections (e.g., HCV infection in B-cells) can lead tohyper(lympho)proliferative disorders.

As used herein, the term immunological binding refers to thenon-covalent interactions that occur between an antibody molecule and anantigen for which the antibody is specific. It also refers to suchinteractions that occur between an antibody in its bound state to anantigen and an Fc receptor in an effector cell. The strength or affinityof immunological binding interactions can be expressed in terms of thedissociation constant (K_(D)) of the interaction, wherein a smallerK_(D) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the on (k_(on)) and the off(k_(off)) rate constants can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of k_(off)/k_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constant K_(D)(Davies et. al., 1990, Ann. Rev. Biochem. 59: 439).

The term “in vitro scanning saturation mutagenesis” (SSM; Monju™) refersto a novel antibody engineering procedure, analogous to somatichypermutation in vivo, for exploring in vitro antibody affinityevolution. An amino acid residue of interest in a protein sequence ismutated to nineteen other possible substitutions, and its effect on thestructure and function of the protein analyzed. Interesting singlemutants can be used as a starting point for subsequent rounds of SSM atother sites, so that multiple mutations with synergistic effects onbinding may be identified. This same sequential mutation approach shouldbe useful to optimize properties such as affinity, potency, efficacy,altered specificity, reduced immunogenicity, and removal of proteolyticcleavage sites (Burks et. al., 1997, Proc. Natl. Acad. Sci. USA 94:412;Chen et. al., 1999, Prot. Eng. 12:349; U.S. Pat. No. 6,180,341).

The term “specifically binds to a protein” refers to a binding reaction,which is determinative of the presence of the protein in the presence ofa heterogeneous population of proteins. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to a protein under suchconditions may require an antibody that is selected for its specificityfor a particular protein (Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Publications, New York, N.Y. (1988)).

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear sequence of amino acidresidues by peptide bonds between the alpha amino and carboxyl Groups ofadjacent residues. The amino acid residues are in many embodiments inthe natural L-isomeric form. However, residues in the D-isomeric formcan be substituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide.

The term “binding polypeptide” refers to a polypeptide that specificallybinds to a target molecule (for example, a cell receptor) in a manneranalogous to the binding of an antibody to an antigen. Bindingpolypeptides can be derived from antibody genes or fragments of antibodygenes. Thus, Fc fragment that binds to Fc receptor is an example of abinding polypeptide.

The substitutions, deletions, inversions, and/or insertions of aminoacids in an antibody (e.g., in an Fc variant antibody) will occur inregions not essential to antigen binding. The identification ofessential and non-essential amino acids in the antibody can be achievedby methods known in the art, such as by site-directed mutagenesis (forexample, SSM) and AlaScan analysis (Moffison et. al., 2001, Chem. Biol.5:302-307). Essential amino acids have to be maintained or replaced byconservative substitutions in the variants. Non-essential amino acidscan be deleted, or replaced by a spacer or by conservative ornon-conservative substitutions.

Antibody variants can be obtained by substitution of any of the aminoacids present in the antibody. For example, Fc variant antibodies can beobtained by substitution of any of the amino acids present in the Fcfragment. As can be appreciated, there are positions in the sequencethat are more tolerant to substitutions than others, and somesubstitutions can improve the binding activity of the parent antibody.The amino acids that are essential should either be identical to theamino acids present in the parent antibody, or substituted byconservative substitutions. The amino acids that are non-essential canbe identical to those in the parent antibody, or can be substituted byconservative or non-conservative substitutions, and/or can be deleted.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Where the side-chain of the amino acid to be replaced is either polar orhydrophobic, the conservative substitution should be with a naturally ornon-naturally occurring amino acid that is also polar or hydrophobic.Conservative amino acid substitutions generally involve substitution ofone amino acid for another amino acid with similar chemical properties(e.g., charge or hydrophobicity). The following six Groups each containamino acids that are typical conservative substitutions for one another:(1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D),Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R),Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative substitution is a substitution in which thesubstituting amino acid (naturally or non-naturally occurring) hassignificantly different size, configuration and/or electronic propertiescompared to the amino acid being substituted. Thus, the side chain ofthe substituting amino acid can be significantly lower (or smaller) thanthe side chain of the native amino acid being substituted and/or canhave functional groups with significantly different electronicproperties than the amino acid being substituted.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et. al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et.al., 1985, J. Biol. Chem. 260:2605). The term nucleic acid is usedinterchangeably with gene, cDNA, and the mRNA encoded by a gene.

The term “heterologous nucleic acid” refers to a nucleic acid that isnot native to the cell in which it is found.

The term “detectable label” refers to any material having a detectablephysical or chemical property. Such detectable labels have been wellestablished in the field of immunoassays. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present disclosure include magnetic beads (for example, DYNABEADS™)fluorescent dyes (example, fluorescein isothiocyanate, rhodamine),enzymes (example, β-galactosidase, chloramphenicol acetyltransferase,horse radish peroxidase, alkaline phosphatase and others, commonly usedas detectable enzymes, either as marker gene products or in anenzyme-linked immunosorbent assay (ELISA). Those detectable labels thatcan be encoded by nucleic acids are referred to as ‘reporter genes’ or‘reporter gene products’.

Functional or active regions of the antibody or antibody fragment may beidentified and/or improved by mutagenesis of a specific region of theprotein, followed by expression and testing of the expressedpolypeptide. For example, amino acid sequence variants of antibodies orantibody fragments can be generated and those that display equivalent orimproved affinity for antigen can be identified using standardtechniques and/or those described herein. One such example is generationof an Fc variant region with an improved affinity for FcγRIIIAcomprising F/F¹⁵⁸ allele. Methods for generating amino acid sequencevariants are readily apparent to a skilled practitioner in the art andcan include site-specific mutagenesis, directed evolution technologies(U.S. Pat. No. 6,180,341) or random mutagenesis (e.g., by PCR) of thenucleic acid encoding the antibody or antibody fragment (Zoller, M. J.,1992, Curr. Opinion in Biotechnol. 3:348-354). Both naturally occurringand non-naturally occurring amino acids may be used to generate aminoacid sequence variants of the antibodies and antibody fragments of thedisclosure.

The “percentage of sequence identity” or “sequence identity” isdetermined by comparing two optimally aligned sequences or subsequencesover a comparison window or span, wherein the portion of thepolynucleotide sequence in the comparison window may optionally compriseadditions or deletions (i.e., gaps) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical residue(e.g., nucleic acid base or amino acid residue) occurs in both sequencesto yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity. Percentage sequence identity when calculated using theprograms GAP or BESTFIT (See, e.g., below) is calculated using defaultgap weights.

Sequences corresponding to the antibodies in the present disclosure maycomprise at least about 70% sequence identity to the sequence of theantibody deposited in GenBank, preferably about 75%, 80% or 85% or morepreferably, about 90% or 95% or more identity. Homology or identity isdetermined by BLAST (Basic Local Alignment Search Tool) analysis usingthe algorithm employed by the programs blastp, blastn, blastx, tblastnand tblastx (Karlin et al., 1990, Proc Natl Acad Sci USA 87:2264-2268;and Altschul, 1993, J Mol Evol 36:290-300) which are tailored forsequence similarity searching. The approach used by the BLAST program isfirst to consider similar segments between a query sequence and adatabase sequence, then to evaluate the statistical significance of allmatches that are identified and finally to summarize only those matcheswhich satisfy a preselected threshold of significance. For a discussionof basic issues in similarity searching of sequence databases, See,e.g., Altschul et al., 1994, Nat Genet. 6:119-129). The searchparameters for histogram, descriptions, alignments, expect (i.e., thestatistical significance threshold for reporting matches againstdatabase sequences), cutoff, matrix and filter are at the defaultsettings. The default scoring matrix used by blastp, blastx, tblastn,and tblastx is the BLOSUM62 matrix (Henikoff et al., 1992, Proc NatlAcad Sci USA 89:10915-10919). Four blastn parameters were adjusted asfollows: Q=10 (gap creation penalty); R=10 (gap extension penalty);wink=−1 (generates word hits at every wink^(th) position along thequery); and gapw=16 (sets the window width within which gappedalignments are generated). The equivalent Blastp parameter settings wereQ=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,available in the GCG package version 10.0, uses DNA parameters GAP=50(gap creation penalty) and LEN=3 (gap extension penalty) and theequivalent settings in protein comparisons are GAP=8 and LEN=2.

DETAILED DESCRIPTION

Methods and compositions are provided for determining whether a subjectsuffering from a neoplastic condition is responsive to an antineoplastictherapy, such as antibody therapy, e.g., Rituximab therapy. Inpracticing the subject methods, the subject is genotyped to determinewhether the subject has at least one favorable FcγR polymorphism, e.g.,the H/H¹³¹ genotype in FcγRIIA or the V/V¹⁵¹ genotype in FcγRIIIA. Inaddition, reagents, devices and kits thereof that find use in practicingthe subject methods are provided.

Methods and compositions are provided for determining whether a subjectsuffering from a neoplastic condition is responsive to an antineoplastictherapy, such as antibody therapy, e.g., rituximab therapy. Inpracticing the subject methods, the subject is genotyped to determinewhether the subject has at least one favorable FcγR polymorphism, e.g.,the H/H¹³¹ genotype in FcγRIIA or the V/V¹⁵⁸ genotype in FcγRIIIA. Inaddition, reagents, devices and kits thereof, that find use inpracticing the subject methods are provided.

Methods are provided for determining the degree of responsiveness that asubject having an ADCC-treatable disease or disorder will have to anantibody therapy for the disease or disorder by genotyping the subjectfor an FcγRIIA polymorphism to obtain a first result, genotyping thesubject for an FcγRIIIA polymorphism to obtain a second result,assigning the subject to one of more than three categories of treatmentresponse and/or employing the first and second results to determine thedegree of the responsiveness of the subject to the antibody therapy.

For example, the FcγRIIA polymorphism can be the H/R¹³¹ polymorphism andthe FcγRIIIA polymorphism can be the V/F¹⁵⁸ polymorphism.

In some embodiments, the presence of both a H/H¹³¹ genotype and a V/V¹⁵⁸genotype indicates a high degree of treatment response to the antibodytherapy. In other embodiments, the identification of i) a H/H¹³¹genotype and ii) a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype indicates an intermediatedegree of treatment response to the antibody therapy. In yet anotherembodiment, the identification of i) a V/V¹⁵⁸ genotype and ii) a H/R¹³¹or a R/R¹³¹ genotype indicates an intermediate degree of treatmentresponse to the antibody therapy. In some embodiments, theidentification of: i) a V/F¹⁵⁸ genotype and a H/R¹³¹ genotype; ii) a 158V/F genotype and a R/R¹³¹ genotype; iii) a F/F¹⁵⁸ genotype and a H/R¹³¹genotype; or iv) a F/F¹⁵⁸ genotype and a R/R¹³¹ genotype indicates a lowdegree of treatment response to the antibody therapy.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In other embodiments, the ADCC-treatable disease ordisorder is a neoplastic disease. In another embodiment, the neoplasticdisease is non-Hodgkin's lymphoma (NHL), e.g., follicular lymphoma.

In some embodiments, the antibody therapy includes the use of atherapeutic antibody or mimetic thereof that specifically binds to CD20.In other embodiments, the therapeutic antibody is a monoclonal antibody,e.g., rituximab.

In some embodiments, the methods include developing patient-groupspecific Fc variant antibodies based on the first and second resultssuch that the therapeutic response rate of the patient group to theantibody therapy is improved.

Methods are provided for selecting a specific Fc variant antibodytherapy from a set of two or more Fc variant antibody therapies for usein treating subjects having an ADCC-treatable disease by genotyping thesubjects for an FcγRIIA polymorphism to classify patient population intothree groups (e.g., H/H¹³¹, H/R¹³¹, R/R¹³¹), genotyping the subjects foran FcγRIIIA polymorphism to classify patient population into threegroups (e.g., V/V¹⁵⁸, V/F¹⁵⁸, F/F¹⁵⁸), classifying the subjects intonine patient groups I-IX based on the first and second results, andemploying the first and second results to select a specific Fc variantantibody therapy for the patient group such that the degree of treatmentresponse to antibody therapy in the patient group is improved.

For example, the FcγRIIA polymorphism can be the H/R¹³¹ polymorphism andthe FcγRIIIA polymorphism can be the V/F¹⁵⁸ polymorphism.

In some embodiments, the genotyping may identify a H/H¹³¹ genotype and aV/V¹⁵⁸ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/H¹³¹ allele and an FcγRIIIA including aV/V¹⁵⁸ allele.

In other embodiments, the genotyping may identify a H/H¹³¹ genotype anda V/F¹⁵⁸ genotype. In this embodiment, the Fc variant antibody can beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/H¹³¹ allele and an FcγRIIIA including aV/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/H¹³¹ allele and an FcγRIIIA including aF/F¹⁵⁸ allele.

In other embodiments, the genotyping may identify a V/F¹⁵⁸ genotype anda H/R¹³¹ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/R¹³¹ allele and an FcγRIIIA including aV/F¹⁵⁸ allele.

In other embodiments, the genotyping may identify a V/F¹⁵⁸ genotype anda R/R¹³¹ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a R/R¹³¹ allele and an FcγRIIIA including aV/F¹⁵⁸ allele.

In other embodiments, the genotyping may identify a F/F¹⁵⁸ genotype anda H/R¹³¹ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/R¹³¹ allele and an FcγRIIIA including aF/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a F/F¹⁵⁸ genotype and aR/R¹³¹ genotype. In this embodiment, the Fc variant antibody can beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a R/R¹³¹ and an FcγRIIIA including a F/F¹⁵⁸allele

In other embodiments, the genotyping may identify a V/V¹⁵⁸ genotype anda H/R¹³¹ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a H/R¹³¹ allele and an FcγRIIIA including aV/V¹⁵⁸ allele.

In other embodiments, the genotyping may identify a V/V¹⁵⁸ genotype anda R/R¹³¹ genotype. In this embodiment, the Fc variant antibody may beselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA including a R/R¹³¹ allele and an FcγRIIIA including aV/V¹⁵⁸ allele.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder is a neoplastic disease. In other embodiments, the neoplasticdisease is non-Hodgkin's lymphoma (NHL), e.g., follicular lymphoma.

In some embodiments, the antibody therapy includes the use of atherapeutic antibody or mimetic thereof that specifically binds to CD20.In other embodiments, the therapeutic antibody is a monoclonal antibody.

In other embodiments, the monoclonal antibody may include one or moreamino acid substitutions compared to rituximab, wherein the one or moreamino acid substitutions provide for enhanced binding and/or in vitroADCC function to at least one of an FcγRIIA comprising H/R¹³¹ or R/R¹³¹alleles, and an FcγRIIIA comprising V/F¹⁵⁸ or F/F¹⁵⁸ alleles.

Methods are also provided for making a set of related antibodies bymodifying the amino acid sequence of at least one Fc region amino acidresidue in a parent antibody, such that the modified Fc region exhibitsenhanced binding affinity to at least one Fc receptor encoded by an Fcreceptor gene of a first genotype, compared to the Fc binding affinityof the parent antibody, to generate a first Fc variant antibody; and/ormodifying at least one Fc region amino acid residue in a parentantibody, such that the modified constant region exhibits enhancedbinding affinity to at least one Fc receptor encoded by an Fc receptorgene of a second genotype, compared to the Fc binding affinity of theparent antibody, to generate a second Fc variant antibody, wherein thefirst and second Fc variant antibodies have the same antigenspecificity.

For example, the first and the second Fc variant antibodies can have oneor more amino acid residue modifications in one or more locations of alower hinge region, a CH2 domain, and/or a CH3 domain.

In some embodiments, the wild-type antibody is a therapeutic antibodyused in therapy of an ADCC-treatable disease or disorder. In someembodiments, the wild-type antibody is a therapeutic antibody used intherapy of a neoplastic disease. In another embodiment, the neoplasticdisease is non-Hodgkin's lymphoma (NHL), e.g., follicular lymphoma.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a viral infection, aparasitic infection, or an allograft rejection.

In some embodiments, the first and second Fc variant antibodies bindspecifically binds to CD20. In some embodiments, the first and second Fcvariant antibodies are monoclonal antibodies, e.g., rituximab.

Also provided are kits for use in determining responsiveness to anantibody therapy in a patient which include an element for genotyping asample to identify an FcγRIIA polymorphism, an element for genotypingthe sample to identify an FcγRIIIA polymorphism, or an element forgenotyping the sample to identify an FcγRIIIA polymorphism and anFcγRIIA, and a reference that correlates a genotype with predictedtherapeutic response to a therapeutic antibody.

In some embodiments, the FcγRIIA genotype may be a H/H¹³¹ genotype andthe FcγRIIIA genotype may be a V/V¹⁵⁸ genotype. In this embodiment, thereference indicates a high degree of responsiveness to the therapeuticantibody.

In other embodiments, the FcγRIIA genotype may be a H/H¹³¹ genotype andthe FcγRIIIA genotype may be a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype. In thisembodiment, the reference indicates an intermediate degree ofresponsiveness to the therapeutic antibody.

In another embodiment, the FcγRIIIA genotype is a V/V¹⁵⁸ genotype andthe FcγRIIA genotype is a H/R¹³¹ or a R/R¹³¹ genotype. In thisembodiment, the reference indicates an intermediate degree ofresponsiveness to the reference therapeutic antibody.

In some embodiments, the reference may indicate choosing an Fc variantantibody that exhibits enhanced binding to an FcγRIIA and/or an FcγRIIIAthat exhibits enhanced in vitro ADCC function.

In some embodiments, the genotype is a V/F¹⁵⁸ genotype and a H/R¹³¹genotype, a V/F¹⁵⁸ genotype and a R/R¹³¹ genotype, a F/F¹⁵⁸ genotype anda H/R¹³¹ genotype, or a F/F¹⁵⁸ genotype and a R/R¹³¹* In thisembodiment, the reference indicates a low degree of responsiveness tothe therapeutic antibody.

In some embodiments, the therapeutic antibody is used for treating anADCC-treatable disease or disorder. In some embodiments, theADCC-treatable disease or disorder may be a neoplastic disease, anautoimmune disease, a microbial infection, or an allograft rejection.

Methods are provided for of treating an ADCC-treatable disease ordisorder in an individual by determining a category of therapeuticresponsiveness to an antibody therapy by genotyping the individual foran FcγRIIA polymorphism and an FcγRIIIA polymorphism, wherein theFcγRIIA polymorphism and the FcγRIIIA polymorphism together indicate adegree of therapeutic responsiveness; selecting an antibody from a setof related antibodies, wherein members of the set of related antibodieshave the same antigen binding specificity, and wherein the members ofthe set of related antibodies differ in binding affinity to an FcγRIIAand/or an FcγRIIIA and/or differ in in vitro ADCC function; andadministering an effective amount of the antibody to the individual.

In some embodiments, the genotyping may identify a H/H¹³¹ genotype and aV/V¹⁵⁸ genotype. In this embodiment, the antibody is selected forbinding to at least one of an FcγRIIA having a H/H¹³¹ allele and anFcγRIIIA having a V/V¹⁵⁸ allele.

In other embodiments, the genotyping may identify an H/H¹³¹ genotype anda V/F¹⁵⁸ or a F/F¹⁵⁸ genotype. In this embodiment, the antibody can beselected for binding to at least one of an FcγRIIA having a His¹³¹ andan FcγRIIIA having a Val¹⁵⁸ or F¹⁵⁸.

In another embodiment, the genotyping may identify a V/F¹⁵⁸ genotype anda H/R¹³¹ genotype; a V/F¹⁵⁸ genotype and a R/R¹³¹ genotype; a F/F¹⁵⁸genotype and a H/R¹³¹ genotype; or a F/F¹⁵⁸ genotype and a R/R¹³¹genotype. In this embodiment, the antibody may be selected for bindingto at least one of an FcγRIIA having a Arg¹³¹ and an FcγRIIIA having aPhe¹⁵⁸.

In other embodiments, the genotyping may identify a H/H¹³¹ genotype anda V/F¹⁵⁸ genotype. In this embodiment, the antibody may be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a H/H¹³¹ allele and an FcγRIIIA havinga V/F¹⁵⁸ allele.

In some embodiment, the genotyping identifies a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype. In this embodiment, the antibody may be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a H/H¹³¹ allele and an FcγRIIIA havinga F/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a V/F¹⁵⁸ genotype and aH/R¹³¹ genotype. In this embodiment, the antibody can be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a H/R¹³¹ allele and an FcγRIIIA havinga V/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a V/F¹⁵⁸ genotype and aR/R¹³¹ genotype. In this embodiment, the antibody can be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a R/R¹³¹ allele and an FcγRIIIA havinga V/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a F/F¹⁵⁸ genotype and aH/R¹³¹ genotype. In this embodiment, the antibody can be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a H/R¹³¹ allele and an FcγRIIIA havinga F/F¹⁵⁸ allele.

In some embodiments, the genotyping may identify a F/F¹⁵⁸ genotype and aR/R¹³¹ genotype. In this embodiment, the antibody can be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a R/R¹³¹ and an FcγRIIIA having aF/F¹⁵⁸ allele.

In other embodiments, the genotyping may identify a V/V¹⁵⁸ genotype anda H/R¹³¹ genotype. In this embodiment, the antibody may be an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a H/R¹³¹ allele and an FcγRIIIA havinga V/V¹⁵⁸ allele.

In some embodiments, the genotyping may identify a V/V¹⁵⁸ genotype and aR/R¹³¹ genotype. In this embodiment, the antibody is an Fc variantantibody selected for enhanced binding and/or in vitro ADCC function toat least one of an FcγRIIA having a R/R¹³¹ allele and an FcγRIIIA havinga V/V¹⁵⁸ allele.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection.

Methods are provided for determining the degree of responsiveness to anantibody-dependent cell-mediated cytotoxicity antibody therapy bygenotyping the subject for two or more Fcγ receptor polymorphisms andemploying the first and second Fcγ receptor polymorphisms to determinethe degree of the responsiveness of the subject to the antibody therapy.

In some embodiments, the Fcγ receptor polymorphisms include an FcγRIIApolymorphism and an FcγRIIIA polymorphism.

For example, an amino acid residue in the Fc region is selectivelysubstituted by nineteen other natural amino acids.

Methods are also provided for generating a set of Fc variant antibodiesby amplifying a nucleic acid comprising a nucleotide sequence encodingan Fc region of an antibody, wherein the amplifying is carried out witha set of primers that encode all nineteen amino acid variants at asingle residue of the Fc region, to generate a set of variant nucleicacids encoding nineteen amino acid substitution variants at the singleresidue of the Fc region; transcribing and translating each of thevariant nucleic acids in vitro, to generate a set of Fc variants; and/orc) selecting from the set an Fc variant having altered FcR bindingactivity compared to a reference Fc, generating a set of selected Fcvariants.

In some embodiments, the methods include determining in vitro ADCCactivity of the selected Fc variant.

In other embodiments, the methods include: d) repeating step (a) at asecond residue of the Fc region, generating a second set of Fc variant;and/or e) repeating steps (b) and (c) on the second set, generating asecond set of selected Fc variants.

In some embodiments, the transcribing and translating is carried outusing a prokaryotic expression system.

In other embodiments, the Fc-encoding nucleotide sequence is under thetranscriptional control of a phage promoter.

In some embodiments, FcR binding activity is assessed using anenzyme-linked immunosorbent assay.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Insome embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In a some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the H/H¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the H/H¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the H/R¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the H/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/V¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theV/V¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the V/F¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theV/F¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for selecting a patient for treatment with anantibody by (a) determining if the patient has (i.) an FcγRIIIA V/V¹⁵⁸genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; or (ii.)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype; (b) selectingthe patient with the F/F¹⁵⁸ genotype, the R/R¹³¹ genotype, or both theF/F¹⁵⁸ genotype, the R/R¹³¹ genotype for treatment with the antibodybased on the genotype determination of steps (i), (ii) or (iii); and (c)administering the antibody to the patient selected in step (b).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA H/H¹³¹genotype; or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA H/H¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA H/H¹³¹genotype; or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA H/H¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, or an FcγRIIAH/H¹³¹ genotype; or both an FcγRIIIA F/F¹⁵⁸ genotype and an FcγRIIAH/H¹³¹ genotype and (b) administering the antibody to the patientselected in step (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype; or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype, or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, an FcγRIIA H/R¹³¹genotype, or both an FcγRIIIA F/F¹⁵⁸ genotype and an FcγRIIA H/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIA R/R¹³¹genotype, or both an FcγRIIIA V/V¹⁵⁸ genotype and an FcγRIIA R/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for treating a patient with an antibody by (a)selecting a patient with an FcγRIIIA V/F¹⁵⁸ genotype, an FcγRIIA R/R¹³¹genotype, or both an FcγRIIIA V/F¹⁵⁸ genotype and an FcγRIIA R/R¹³¹genotype and (b) administering the antibody to the patient selected instep (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for treating a patient with an antibody,comprising: (a) selecting a patient with an FcγRIIIA F/F¹⁵⁸ genotype, anFcγRIIA R/R¹³¹ genotype, or both an FcγRIIIA F/F¹⁵⁸ genotype and anFcγRIIA R/R¹³¹ genotype and (b) administering the antibody to thepatient selected in step (a).

In some embodiments, the patient has a hyperproliferative disorder. Inother embodiments, the hyperproliferative disorder is cancer, e.g.,non-Hodgkin's lymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are also provided for classifying a subject having anADCC-treatable disease or disorder into one of more than threecategories of responsiveness to an antibody therapy by genotypingsubjects for an FcγRIIA polymorphism and an FcγRIIIA polymorphism,wherein the subjects have or had the ADCC-treatable disease or disorderand are or were administered antibody therapy for the disease ordisorder; classifying each subject based on its FcγRIIA polymorphism andFcγRIIIA polymorphism to one of three or more categories ofresponsiveness to the antibody therapy; genotyping the subject for anFcγRIIA polymorphism and an FcγRIIIA polymorphism; identifying agenotype from (a) that is identical to the genotype from the subject instep (c), wherein the subject is classified into a category ofresponsiveness to the antibody therapy for the disease or disordercorresponding with a subject having an identical FcγRIIA polymorphismand an identical FcγRIIIA polymorphism.

In one embodiment, the subjects are classified into one of ninecategories of responsiveness to the antibody therapy.

For example, the FcγRIIA polymorphism may be the H/R¹³¹ polymorphism andthe FcγRIIIA polymorphism may be the V/F¹⁵⁸ polymorphism.

In some embodiments, the presence of both a H/H¹³¹ genotype and a V/V¹⁵⁸genotype indicates a high degree of treatment response to the antibodytherapy.

In other embodiments, the identification of i) a H/H¹³¹ genotype and ii)a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype indicates an intermediate degree oftreatment response to the antibody therapy.

In other embodiments, the identification of i) a V/V¹⁵⁸ genotype and ii)a H/R¹³¹ or a R/R¹³¹ genotype indicates an intermediate degree oftreatment response to the antibody therapy.

In some embodiments, the identification of: i) a V/F¹⁵⁸ genotype and aH/R¹³¹ genotype; ii) a 158 V/F genotype and a R/R¹³¹ genotype; iii) aF/F¹⁵⁸ genotype and a H/R¹³¹ genotype; or iv) a F/F¹⁵⁸ genotype and aR/R¹³¹ genotype indicates a low degree of treatment response to theantibody therapy.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder may be a neoplastic disease. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Methods are provided for determining the degree of responsiveness that asubject having an ADCC-treatable disease or disorder will have to anantibody therapy for the disease or disorder by genotyping the subjectfor an FcγRIIA polymorphism and an FcγRIIIA polymorphism; andidentifying a genotype associated with a particular degree ofresponsiveness to the antibody therapy from a reference that isidentical to the genotype from the test subject, wherein the testsubject is determined to have a degree of responsiveness to the antibodytherapy for the disease or disorder corresponding to the level ofresponsiveness associated with the reference having an identical FcγRIIApolymorphism and an identical FcγRIIIA polymorphism.

In some embodiments, the subjects may be classified into one of ninecategories of responsiveness to the antibody therapy.

In some embodiments, the FcγRIIA polymorphism is the H/R¹³¹ polymorphismand the FcγRIIIA polymorphism is the V/F¹⁵⁸ polymorphism. In otherembodiments, the presence of both a H/H¹³¹ genotype and a V/V¹⁵⁸genotype indicates a high degree of treatment response to the antibodytherapy. In other embodiments, the identification of i) a H/H¹³¹genotype and ii) a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype indicates an intermediatedegree of treatment response to the antibody therapy. In otherembodiments, the identification of i) a V/V¹⁵⁸ genotype and ii) a H/R¹³¹or a R/R¹³¹ genotype indicates an intermediate degree of treatmentresponse to the antibody therapy. In yet other embodiments, theidentification of: i) a V/F¹⁵⁸ genotype and a H/R¹³¹ genotype; ii) a 158V/F genotype and a R/R¹³¹ genotype; iii) a F/F¹⁵⁸ genotype and a H/R¹³¹genotype; or iv) a F/F¹⁵⁸ genotype and a R/R¹³¹ genotype indicates a lowdegree of treatment response to the antibody therapy.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder may be a neoplastic disease. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

For example, the reference may be an index including the genotypes forsubjects that had an ADCC-treatable disease or disorder and wereadministered antibody therapy for the disease or disorder and whereinthe subjects were classified into one of more than three categories ofresponsiveness to the antibody therapy.

Methods are also provided for determining the degree of responsivenessthat a test subject having an ADCC-treatable disease or disorder willhave to an antibody therapy for the disease or disorder by (a)genotyping subjects for an FcγRIIA polymorphism and an FcγRIIIApolymorphism, wherein the subjects have or had the ADCC-treatabledisease or disorder and are or were administered antibody therapy forthe disease or disorder; (b) classifying each subject based on itsFcγRIIA polymorphism and FcγRIIIA polymorphism to one of more than threecategories of responsiveness to the antibody therapy; (c) genotyping thetest subject for an FcγRIIA polymorphism and an FcγRIIIA polymorphism;and (d) identifying a genotype from (a) that is identical to thegenotype from the test subject in step (c), wherein the test subject isdetermined to have a degree of responsiveness to the antibody therapyfor the disease or disorder corresponding to the level of responsivenessassociated with a subject having an identical FcγRIIA polymorphism andan identical FcγRIIIA polymorphism.

For example, the subjects may be classified into one of nine categoriesof responsiveness to the antibody therapy.

For example, the FcγRIIA polymorphism may be the H/R¹³¹ polymorphism andthe FcγRIIIA polymorphism may be the V/F¹⁵⁸ polymorphism.

In some embodiments, the presence of both a H/H¹³¹ genotype and a V/V¹⁵⁸genotype indicates a high degree of treatment response to the antibodytherapy. In other embodiments, the identification of i) a H/H¹³¹genotype and ii) a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype indicates an intermediatedegree of treatment response to the antibody therapy. In otherembodiments, the identification of i) a V/V¹⁵⁸ genotype and ii) a H/R¹³¹or a R/R¹³¹ genotype indicates an intermediate degree of treatmentresponse to the antibody therapy. In other embodiments, theidentification of: i) a V/F¹⁵⁸ genotype and a H/R¹³¹ genotype; ii) a 158V/F genotype and a R/R¹³¹ genotype; iii) a F/F¹⁵⁸ genotype and a H/R¹³¹genotype; or iv) a F/F¹⁵⁸ genotype and a R/R¹³¹ genotype indicates a lowdegree of treatment response to the antibody therapy.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder may be a neoplastic disease. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the antibody is a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

Also provided are kits for use in determining responsiveness to anantibody therapy in a patient which include an element for genotypingthe sample to identify an FcγRIIA polymorphism; an element forgenotyping the sample to identify an FcγRIIIA polymorphism; and areference that correlates a genotype in the patient with one of morethan three predicted therapeutic responses to the antibody therapy.

For example, the reference may correlate a genotype in the patient withone of nine predicted therapeutic responses to the antibody therapy.

In some embodiments, the FcγRIIA genotype is a H/H¹³¹ genotype, whereinthe FcγRIIIA genotype is a V/V¹⁵⁸ genotype, and wherein the referenceindicates a high degree of responsiveness to the therapeutic antibody.

In other embodiments, the FcγRIIA genotype is a H/H¹³¹ genotype, whereinthe FcγRIIIA genotype is a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype, and wherein thereference indicates an intermediate degree of responsiveness to thetherapeutic antibody.

In some embodiments, the FcγRIIIA genotype is a V/V¹⁵⁸ genotype, whereinthe FcγRIIA genotype is a H/R¹³¹ or a R/R¹³¹ genotype, and wherein thereference indicates an intermediate degree of responsiveness to thereference therapeutic antibody.

In other embodiments, the genotype is i) a V/F¹⁵⁸ genotype and a H/R¹³¹genotype; ii) a V/F¹⁵⁸ genotype and a R/R¹³¹ genotype; iii) a F/F¹⁵⁸genotype and a H/R¹³¹ genotype; or iv) a F/F¹⁵⁸ genotype and a R/R¹³¹,and wherein the reference indicates a low degree of responsiveness tothe therapeutic antibody.

In some embodiments, the reference indicates choosing an variantantibody that exhibits enhanced binding to an FcγRIIA and/or an FcγRIIIAand/or that exhibits enhanced in vitro ADCC function.

In some embodiments, the therapeutic antibody is used for treating anADCC-treatable disease or disorder. In some embodiments, theADCC-treatable disease or disorder may be a neoplastic disease, anautoimmune disease, a microbial infection, or an allograft rejection. Insome embodiments, the neoplastic disease is non-Hodgkin's lymphoma(NHL), e.g., follicular lymphoma.

Methods are provided for selecting a specific variant antibody therapyfrom a set of two or more variant antibody therapies for use intreatment of subjects having an ADCC-treatable disease by genotyping thesubjects for an FcγRIIA polymorphism and an FcγRIIIA polymorphism,classifying the subjects into one of more than three categories ofresponsiveness based on their FcγRIIA polymorphism and their FcγRIIIApolymorphism, and selecting a specific variant antibody therapy for thesubjects such that the degree of responsiveness to the antibody therapyin the subjects is improved from the degree of responsiveness obtainedwith another variant antibody.

In some embodiments, the subjects are classified into one of ninecategories of responsiveness to the antibody therapy.

For example, the FcγRIIA polymorphism can be the H/R¹³¹ polymorphism andthe FcγRIIIA polymorphism can be the V/F¹⁵⁸ polymorphism.

In some embodiments, the presence of both a H/H¹³¹ genotype and a V/V¹⁵⁸genotype indicates a high degree of treatment response to the antibodytherapy. In other embodiments, the identification of i) a H/H¹³¹genotype and ii) a V/F¹⁵⁸ or a F/F¹⁵⁸ genotype indicates an intermediatedegree of treatment response to the antibody therapy. In otherembodiments, the identification of i) a V/V¹⁵⁸ genotype and ii) a H/R¹³¹or a R/R¹³¹ genotype indicates an intermediate degree of treatmentresponse to the antibody therapy. In yet other embodiments, theidentification of: i) a V/F¹⁵⁸ genotype and a H/R¹³¹ genotype; ii) a 158V/F genotype and a R/R¹³¹ genotype; iii) a F/F¹⁵⁸ genotype and a H/R¹³¹genotype; or iv) a F/F¹⁵⁸ genotype and a R/R¹³¹ genotype indicates a lowdegree of treatment response to the antibody therapy.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder may be a neoplastic disease. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the antibody may be a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

For example, the monoclonal antibody may include one or more amino acidsubstitutions compared to Rituximab, wherein the one or more amino acidsubstitutions provide for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising H/R¹³¹ or R/R¹³¹ alleles, andan FcγRIIIA comprising V/F¹⁵⁸ or F/F¹⁵⁸ alleles.

Methods are also provided for treating an ADCC-treatable disease ordisorder in a subject by genotyping the subject for an FcγRIIApolymorphism and an FcγRIIIA polymorphism, classifying the subject intoone of more than three categories of therapeutic responsiveness to anantibody therapy based on the FcγRIIA polymorphism and the FcγRIIIApolymorphism, selecting an antibody with a preferred degree oftherapeutic responsiveness from a set of related antibodies, whereinmembers of the set of related antibodies have the same antigen bindingspecificity, and wherein the members of the set of related antibodiesdiffer in binding affinity to an FcγRIIA and/or an FcγRIIIA and/ordiffer in in vitro ADCC function, and administering a therapeuticallyeffective amount of the antibody to the subject, wherein, the antibodytreats the ADCC-treatable disease or disorder in the subject.

In some embodiments, the subject is classified into one of ninecategories of therapeutic responsiveness.

For example, the FcγRIIA polymorphism may be the H/R¹³¹ polymorphism,and the FcγRIIIA polymorphism may be the V/F¹⁵⁸ polymorphism.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aV/V¹⁵⁸ genotype, and the antibody is selected for binding to at leastone of an FcγRIIA comprising H/H¹³¹ allele and an FcγRIIIA comprisingV/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aV/F¹⁵⁸ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising F/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising V/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising V/V¹⁵⁸ allele.

In other embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising V/F¹⁵⁸ allele.

In other embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a F/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising F/F¹⁵⁸ allele.

In other embodiments, the genotyping identifies a F/F¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ and an FcγRIIIA comprising F/F¹⁵⁸ allele.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the ADCC-treatable disease ordisorder may be a neoplastic disease. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the antibody may be a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

In another embodiment, the monoclonal antibody includes one or moreamino acid substitutions compared to Rituximab, wherein the one or moreamino acid substitutions provide for enhanced binding and/or in vitroADCC function to at least one of an FcγRIIA comprising H/R¹³¹ or R/R¹³¹alleles, and an FcγRIIIA comprising V/F¹⁵⁸ or F/F¹⁵⁸ alleles.

Methods are provided for making a set of related antibodies capable ofmodulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorderby modifying the amino acid sequence of at least one amino acid residuein a parent antibody, such that the modified parent antibody exhibitsenhanced binding affinity to at least one Fc receptor encoded by an Fcreceptor gene of a first genotype, compared to the Fc binding affinityof the parent antibody, to generate a first variant antibody; andmodifying at least one amino acid residue in a parent antibody, suchthat the modified parent antibody exhibits enhanced binding affinity toat least one Fc receptor encoded by an Fc receptor gene of a secondgenotype, compared to the Fc binding affinity of the parent antibody, togenerate a second variant antibody, wherein the first and second variantantibodies have the same antigen specificity and are capable ofmodulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorder.

For example, the first and the second variant antibodies may include oneor more amino acid residue modifications in one or more locations of alower hinge region, a CH2 domain, and/or a CH3 domain.

In some embodiments, the parent antibody may be a therapeutic antibodyused in therapy of an ADCC-treatable disease or disorder.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a viral infection, aparasitic infection, or an allograft rejection. In some embodiments, theneoplastic disease is non-Hodgkin's lymphoma (NHL), e.g., follicularlymphoma.

In some embodiments, the parent antibody is a therapeutic antibody,e.g., RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®,AVASTIN®, REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®,TYSABRI®, MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® orZEVALIN®.

In other embodiments, the parent antibody is modified in its Fc domain.In another embodiment, the parent antibody is modified in its CDR.

Methods are provided for generating a set of variant antibodies capableof modulating the responsiveness of a subject having an ADCC-treatabledisease or disorder to an antibody therapy for the disease or disorderby amplifying a nucleic acid comprising a nucleotide sequence encoding aregion of an antibody, wherein the amplifying is carried out with a setof primers that encode all nineteen amino acid variants at a singleresidue of the region, to generate a set of variant nucleic acidsencoding nineteen amino acid substitution variants at the single residueof the region, transcribing and translating each of the variant nucleicacids in vitro, to generate a set of variants, and/or selecting from theset an variant having altered FcR binding activity compared to areference region, generating a set of selected variants, wherein thefirst and second variant antibodies have the same antigen specificityand are capable of modulating the responsiveness of a subject having anADCC-treatable disease or disorder to an antibody therapy for thedisease or disorder. In some embodiments, the method includesdetermining in vitro ADCC activity of the selected variant.

In some embodiments, the methods include, repeating step amplifying anucleic acid comprising a nucleotide sequence encoding a region of anantibody, wherein the amplifying is carried out with a set of primersthat encode all nineteen amino acid variants at a single residue of theregion at a second residue of the region, generating a second set ofvariants and generating a second set of selected variants.

In some embodiments, the transcribing and translating are carried outusing a prokaryotic expression system. For example, the region-encodingnucleotide sequence may be under the transcriptional control of a phagepromoter.

In some embodiments, FcR binding activity is assessed using anenzyme-linked immunosorbent assay.

In some embodiments, the parent antibody is modified in its Fc domain.In other embodiments, the parent antibody is modified in its CDR.

Methods are also provided for modulating the responsiveness of a subjecthaving an ADCC-treatable disease or disorder to an antibody therapy forthe disease or disorder by genotyping the subject for an FcγRIIApolymorphism and an FcγRIIIA polymorphism, classifying the subject intoone of more than three categories of therapeutic responsiveness to anantibody therapy based on the FcγRIIA polymorphism and the FcγRIIIApolymorphism, selecting an antibody from a set of related antibodies,wherein members of the set of related antibodies have the same antigenbinding specificity, and wherein the members of the set of relatedantibodies differ in binding affinity to an FcγRIIA and/or an FcγRIIIAand/or differ in in vitro ADCC function, and administering atherapeutically effective amount of the antibody to the subject, whereinthe antibody modulates the responsiveness of the subject having anADCC-treatable disease or disorder to an antibody therapy for thedisease or disorder.

For example, the subject may be classified into one of nine categoriesof therapeutic responsiveness.

In some embodiments, the antibody increases responsiveness to theantibody therapy. In other embodiments, the antibody decreasesresponsiveness to the antibody therapy.

For example, the FcγRIIA polymorphism may be the H/R¹³¹ polymorphism,and the FcγRIIIA polymorphism may be the V/F¹⁵⁸ polymorphism.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aV/V¹⁵⁸ genotype, and the antibody is selected for enhanced binding to atleast one of an FcγRIIA comprising an H/H¹³¹ allele and an FcγRIIIAcomprising an V/V¹⁵⁸ allele.

In other embodiments the genotyping identifies a H/H¹³¹ genotype and aV/F¹⁵⁸ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an V/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising an V/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a F/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies or a F/F¹⁵⁸ genotype anda R/R¹³¹ genotype, and the variant antibody is selected for enhancedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the neoplastic disease may benon-Hodgkin's lymphoma (NHL), e.g., follicular lymphoma.

In some embodiments, the antibody may be a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

For example, the related antibodies may include one or more amino acidsubstitutions compared to Rituximab, wherein the one or more amino acidsubstitutions provide for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising H/R¹³¹ or R/R¹³¹ alleles, andan FcγRIIIA comprising V/F¹⁵⁸ or F/F¹⁵⁸ alleles.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aV/V¹⁵⁸ genotype, and the antibody is selected for decreased binding toat least one of an FcγRIIA comprising H/H¹³¹ allele and an FcγRIIIAcomprising an V/V¹⁵⁸ allele.

In other embodiments, the genotyping identifies a H/H¹³¹ genotype and aV/F¹⁵⁸ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/H¹³¹ allele and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an V/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/V¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising an V/V¹⁵⁸ allele.

In some embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In other embodiments, the genotyping identifies a V/F¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ allele and an FcγRIIIA comprising an V/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a F/F¹⁵⁸ genotype and aH/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising H/R¹³¹ allele and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the genotyping identifies a F/F¹⁵⁸ genotype and aR/R¹³¹ genotype, and the variant antibody is selected for decreasedbinding and/or in vitro ADCC function to at least one of an FcγRIIAcomprising R/R¹³¹ and an FcγRIIIA comprising an F/F¹⁵⁸ allele.

In some embodiments, the ADCC-treatable disease or disorder may be aneoplastic disease, an autoimmune disease, a microbial infection, or anallograft rejection. In some embodiments, the neoplastic disease isnon-Hodgkin's lymphoma (NHL), e.g., follicular lymphoma.

In some embodiments, the antibody may be a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

In some embodiments, the related antibodies include one or more aminoacid substitutions compared to Rituximab, wherein the one or more aminoacid substitutions provide for decreased binding and/or in vitro ADCCfunction to at least one of an FcγRIIA comprising H/R¹³¹ or R/R¹³¹alleles, and an FcγRIIIA comprising V/F¹⁵⁸ or F/F¹⁵⁸ alleles.

Methods are provided for enhancing antibody dependent cell mediatedcytotoxicity (ADCC) activity of an antibody for use in treatment of asubject having an ADCC-treatable disease by genotyping the subject foran FcγRIIA polymorphism and an FcγRIIIA polymorphism, selecting an Fcnucleotide sequence for the antibody that has optimal ADCC for theFcγRIIA polymorphism and FcγRIIIA polymorphism, and modifying theantibody to include the optimal Fc sequence for the subject's genotype,wherein the ADCC activity of the antibody is enhanced by using theoptimal Fc.

For example, the FcγRIIA polymorphism may be the H/R¹³¹ polymorphism,and the FcγRIIIA polymorphism may be the V/F¹⁵⁸ polymorphism.

In some embodiments, the patient may have a hyperproliferative disorder.In other embodiments, the hyperproliferative disorder may be cancer,e.g., non-Hodgkin's lymphoma.

In some embodiments the antibody may be a therapeutic antibody, e.g.,RITUXAN®, CAMPATH®, ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®,REMICADE®, HUMIRA®, ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®,MYLOTARG®, REOPRO®, LUCENTIS®, SOLIRIS®, OKT3®, BEXXAR® or ZEVALIN®.

The genotypes determined by any of the above-disclosed methods mayinclude (i.) an FcγRIIIA V/V¹⁵⁸ genotype, an FcγRIIIA V/F¹⁵⁸ or anFcγRIIIA F/F¹⁵⁸ genotype; or (an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype, or (iii.) an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/H¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/H¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA H/R¹³¹genotype, an FcγRIIIA F/F¹⁵⁸, FcγRIIA H/R¹³¹ genotype, an FcγRIIIAV/V¹⁵⁸, FcγRIIA R/R¹³¹ genotype, an FcγRIIIA V/F¹⁵⁸, FcγRIIA R/R¹³¹genotype or an FcγRIIIA F/F¹⁵⁸, FcγRIIA R/R¹³¹ genotype.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure,representative illustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

As summarized above, the subject disclosure is directed to methods ofdetermining whether a subject suffering from a neoplastic condition isresponsive to a particular therapy, such as antibody therapy, as well asreagents and kits thereof (and devices) for use in practicing thesubject methods. In further describing the disclosure, the subjectmethods are described first, followed by a review of the reagents, kitsand devices for use in practicing the subject methods.

Methods of Determining Whether a Subject Suffering from a NeoplasticCondition is Responsive to a Particular Therapy

Methods are provided for determining whether a patient or subjectsuffering from a neoplastic disease, i.e., hyperproliferative disorder,is responsive to a particular therapy, such as antibody therapy.Hyperproliferative disorders, or malignancies, are conditions in whichthere is unregulated cell growth. The methods of the present disclosureare directed at hyperproliferative disorders and particularly whethersuch a disorder will or will not be responsive to a particularantineoplastic therapy, e.g., antibody therapy. The disorder may becharacterized by the presence or absence of solid tumors.

In certain embodiments, the subject methods are directed to determiningwhether a B-cell hyperproliferative disorder, e.g., NHL, is responsiveto therapeutic antibody therapy. B-cell hyperproliferative disorders arethose disorders that derive from cells in the B cell lineage, typicallyincluding hematopoietic progenitor cells expressing B lineage markers,pro-B cells, pre-B cells, B-cells and memory B cells; and that expressmarkers typically found on such B lineage cells.

Of particular interest are non-Hodgkin's lymphomas (NHLs), which are aheterogeneous group of lymphoproliferative malignancies with differentpatterns of behavior and responses to treatment. Like Hodgkin's disease,NHL usually originates in lymphoid tissues and can spread to otherorgans, however, NHL is much less predictable than Hodgkin's diseaseregarding their responses to therapy and has a far greater predilectionto disseminate to extranodal sites. The NHLs can be divided into 2prognostic groups: the indolent lymphomas and the aggressive lymphomas.Indolent NHL types have a relatively good prognosis, with mediansurvival as long as 10 years, but they usually are not curable inadvanced clinical stages. The aggressive type of NHL has a shorternatural history. A number of these patients can be cured with intensivecombination chemotherapy regimens, but there is a significant number ofrelapses, particularly in the first 2 years after therapy.

Among the NHL are a variety of B-cell neoplasms, including precursorB-lymphoblastic leukemia/lymphoma; peripheral B-cell neoplasms, e.g.,B-cell chronic lymphocytic leukemia; prolymphocytic leukemia; smalllymphocytic lymphoma; mantle cell lymphoma; follicle center celllymphoma; marginal zone B-cell lymphoma; splenic marginal zone lymphoma;hairy cell leukemia; diffuse large B-cell lymphoma; T-cell rich B-celllymphoma, Burkitt's lymphoma; high-grade B-cell lymphoma,(Burkitt-like); etc.

Follicular lymphoma comprises 70% of the indolent lymphomas reported inAmerican and European clinical trials. Most patients with follicularlymphoma are over age 50 and present with widespread disease atdiagnosis. Nodal involvement is most common, often accompanied bysplenic and bone marrow disease. The vast majority of patients arediagnosed with advanced stage follicular lymphoma and are not cured withcurrent therapeutic options, and the rate of relapse is fairlyconsistent over time, even in patients who have achieved completeresponses to treatment. Subtypes include follicular small cleaved cell(grade 1) and follicular mixed small cleaved and large cell (grade 2).Another subtype of interest is follicular large cell (grade 3 or FLC)lymphoma which can be divided into grades 3a and 3b.

Marginal zone lymphomas were previously included among the diffuse smalllymphocytic lymphomas. When marginal zone lymphomas involve the nodes,they are called monocytoid B-cell lymphomas, and when they involveextranodal sites (gastrointestinal tract, thyroid, lung, breast, skin),they are called mucosa-associated lymphatic tissue (MALT) lymphomas.Many patients have a history of autoimmune disease, such as Hashimoto'sthyroiditis or Sjogren's syndrome, or of Helicobacter gastritis. Mostpatients present with stage I or II extranodal disease, which is mostoften in the stomach. When disseminated to lymph nodes, bone marrow, orblood, this entity behaves like other low-grade lymphomas. Large B-celllymphomas of MALT sites are classified and treated as diffuse large celllymphomas.

Splenic marginal zone lymphoma is an indolent lymphoma that is marked bymassive splenomegaly and peripheral blood and bone marrow involvement,usually without adenopathy. This type of lymphoma is otherwise known assplenic lymphoma with villous lymphocytes, an uncommon variant of B-cellchronic lymphocytic leukemia. Management of this entity usually startswith splenectomy which is different than other low-grade lymphomas.If/when the disease progresses after splenectomy, it tends to be managedlike other low grade lymphomas.

Among the aggressive forms of NHL is diffuse large B-cell lymphoma,which is the most common of the non-Hodgkin's lymphomas, comprising 30%of newly-diagnosed cases. Most patients present with rapidly enlargingmasses, often with symptoms both locally and systemically. Relapsesafter treatment are not uncommon, depending on the presence of variousrisk factors. Lymphomatoid granulomatosis is an EBV positive largeB-cell lymphoma with a predominant T-cell background. The histologyshows association with angioinvasion and vasculitis, usually manifestingas pulmonary lesions or paranasal sinus involvement. Patients aremanaged like others with diffuse large cell lymphoma.

Primary mediastinal B-cell lymphoma is a subset of diffuse large celllymphoma characterized by significant fibrosis on histology. Patientsare usually female and young. Patients present with a locally invasiveanterior mediastinal mass which may cause respiratory symptoms orsuperior vena cava syndrome. Therapy and prognosis are the same as forother comparably-staged patients with diffuse large cell lymphoma,except for advanced-stage patients with a pleural effusion, who have anextremely poor prognosis (progression-free survival is less than 20%)whether the effusion is cytologically positive or negative.

Mantle cell lymphoma is found in lymph nodes, the spleen, bone marrow,blood, and sometimes the gastrointestinal system (lymphomatouspolyposis). Mantle cell lymphoma is characterized by CD5-positive mantlezone B cells, a translocation of chromosomes 11 and 14, and anoverexpression of the cyclin D1 protein. The median survival issignificantly shorter (3-5 years) than that of other lymphomas, and thishistology is now considered to be an aggressive lymphoma. A diffusepattern and the blastoid variant have an even more aggressive coursewith shorter survival, while the mantle zone type may have a moreindolent course. Refractoriness to chemotherapy is a usual feature.

Lymphoblastic lymphoma is a very aggressive form of NHL. It often occursin young patients, but not exclusively. It is commonly associated withlarge mediastinal masses and has a high predilection for disseminatingto bone marrow and the central nervous system (CNS). Treatment isusually patterned after that for acute lymphoblastic leukemia (ALL).Since these forms of NHL tend to progress so quickly, combinationchemotherapy is instituted rapidly once the diagnosis has beenconfirmed. Careful review of the pathologic specimens, bone marrowaspirate and biopsy specimen, cerebrospinal fluid cytology, andlymphocyte marker constitute the most important aspects of thepretreatment staging work-up.

Burkitt's lymphoma/diffuse small noncleaved cell lymphoma typicallyinvolves younger patients and represents the most common type ofpediatric non-Hodgkin's lymphoma. These aggressive extranodal B-celllymphomas are characterized by translocation and deregulation of thec-myc gene on chromosome 8. A subgroup of patients with dualtranslocation of c-myc and bcl-2 appear to have an extremely pooroutcome despite aggressive therapy. Treatment of Burkitt'slymphoma/diffuse small noncleaved cell lymphoma involves aggressivemultidrug regimens similar to those used for the advanced-stageaggressive lymphomas.

Patients who undergo transplantation of the heart, lung, liver, kidney,or pancreas usually require life-long immunosuppression. Life-longimmunosuppression may result in post-transplantation lymphoproliferativedisorder (PTLD), which appears as an aggressive lymphoma. Pathologistscan distinguish a polyclonal B-cell hyperplasia from a monoclonal B-celllymphoma; both are almost always associated with EBV. In some cases,usually for the polyclonal forms of the disease, withdrawal ofimmunosuppression results in eradication of the lymphoma. When this isunsuccessful or not feasible, a combination chemotherapy is usuallyused. EBV-negative post-transplantation lymphoproliferative disordersoccur late and have a particularly poor prognosis. Chronic lymphocyticleukemia (CLL) is a disorder of morphologically mature butimmunologically less mature lymphocytes and is manifested by progressiveaccumulation of these cells in the blood, bone marrow, and lymphatictissues. Lymphocyte counts in the blood are usually equal to or higherthan 10,000 per cubic millimeter. At present there is no curativetherapy. CLL occurs primarily in middle-aged and elderly individuals,with increasing frequency in successive decades of life. The clinicalcourse of this disease progresses from an indolent lymphocytosis withoutother evident disease to one of generalized lymphatic enlargement withconcomitant pancytopenia. Complications of pancytopenia, includinghemorrhage and infection, represent a major cause of death in thesepatients. Immunological aberrations, including Coombs-positive hemolyticanemia, immune thrombocytopenia, and depressed immunoglobulin levels mayall complicate the management of CLL. CLL lymphocytes coexpress theB-cell antigens CD19 and CD20 along with the T-cell antigen CD5. CLL Bcells express relatively low levels of surface-membrane immunoglobulin(compared with normal peripheral blood B cells). CLL is diagnosed by anabsolute increase in lymphocytosis and/or bone marrow infiltrationcoupled with the characteristic features of morphology andimmunophenotype.

AIDS-related lymphomas are comprised of a narrow spectrum of histologictypes consisting almost exclusively of B-cell tumors of aggressive type.These include diffuse large cell lymphoma; B-immunoblastic; and smallnon-cleaved, either Burkitt's or Burkitt's like. The HIV-associatedlymphomas can be categorized into: primary central nervous systemlymphoma (PCNSL); systemic lymphoma; and primary effusion lymphoma. Allof these lymphomas differ from non-HIV-related lymphomas in theirmolecular characteristics, presumed mechanism of pathogenesis,treatment, and clinical outcome. All three pathologic types are equallydistributed and represent aggressive disease. In general, the clinicalsetting and response to treatment of patients with AIDS-related lymphomais very different from the non-HIV patients with lymphoma. TheHIV-infected individual with aggressive lymphoma usually presents withadvanced-stage disease that is frequently extranodal. The clinicalcourse is more aggressive, and the disease is both more extensive andless responsive to chemotherapy. Immunodeficiency and cytopenias, commonin these patients at the time of initial presentation, are exacerbatedby the administration of chemotherapy. Therefore, treatment of themalignancy increases the risk of opportunistic infections that, in turn,further compromise the delivery of adequate treatment.

Acute lymphocytic leukemia (ALL) generally has an aggressive course,depending in part on the presence of the Philadelphia (Ph) chromosome.Patients with Ph chromosome-positive ALL are rarely cured withchemotherapy. Many patients who have molecular evidence of the bcr-ablfusion gene, which characterizes the Ph chromosome, have no evidence ofthe abnormal chromosome by cytogenetics.

Although the methods of the disclosure are primarily applied to NHL, insome cases treatment may be used in cases of Hodgkin's lymphoma, whichis a lymphoma characterized by a pleomorphic lymphocytic infiltrate withmalignant multinucleated giant cells. Most cases of Hodgkin's diseaseprobably arise from germinal center B cells that are unable tosynthesize immunoglobulin. The majority of cases in developing countriesand about one third of those in the United States are associated withthe presence of Epstein-Barr virus in the Reed-Sternberg cells.Treatment strategies depend on a number of factors including thepresence of B symptoms, the histologic subtype, gender, and sexualmaturity. To date there are several published studies demonstrating theeffectiveness of Rituxan for CD20-positive Hodgkin's disease,particularly the lymphocyte predominant variant.

Other neoplastic disease conditions whose responsiveness to antibodytherapy can be evaluated according to the subject methods include, butare not limited to: colorectal cancer, non-small cell lung cancer, smallcell lung cancer, ovarian cancer, breast cancer, head and neck cancer,renal cell carcinoma, and the like.

As summarized above, the subject methods may be used to evaluate theresponsiveness of a subject to a given antineoplastic therapy.Antineoplastic therapies of interest include, but are not limited to:chemotherapy, radiation therapy, antibody therapy, etc.

By therapeutic antibody therapy is meant a treatment protocol or regimenthat includes administration of a therapeutic antibody agent.Representative therapeutic antibody agents specifically bind to antigenspresent on B cells, particularly hyperproliferative B cells, e.g., Blineage lymphomas and leukemias, and the like. The term “antibody” isused in the broadest sense and specifically covers monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments, and Fc:fusion proteins so long as they exhibit the desiredbiological activity. Fragments comprise a portion of a full-lengthantibody, generally the antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.In some aspects of the disclosure, a combination of one or moreantibodies with different specificities, either for epitopes of a singleantigen, or for multiple antigens, may be used.

Markers that are specifically found on B-cells include CD45R, which isan exon specific epitope found on essentially all B-cells, and ismaintained throughout B cell development (Coffman et al., 1982, Immunol.Rev. 69:5-23). The B-cell markers CD19, CD20; CD22; CD23 are selectivelyexpressed on B-cells and have been associated with B-cell malignancies(Kalil and Cheson, 2000, Drugs Aging 16(1):9-27; U.S. Pat. No.6,183,744, herein incorporated by reference). Surface immunoglobulin,including epitopes present on the constant regions or idiotypicdeterminants, is a specific marker for B-cells and has been utilized inimmunotherapy (Caspar et al., 1997, Blood 90(9):3699-706). The MB-1antigen is found on all normal immunoglobulin (Ig)-expressing cells, butnot on T-cells, thymocytes, granulocytes, or platelets, and expressed byvirtually all Ig-expressing B-cell tumors (Link et al., 1986, J.Immunol. 137(9):3013-8). Other B cell antigens of interest known to beexpressed, for example, on non-Hodgkin's lymphomas, are Muc-1; B5; BB1;and T9 (Freedman et al., 1987, Leukemia 1(1):9-15).

Of particular interest is the CD20 antigen, also known as “Bp35”. (Notethat CD20 was called B1 early in the course of research on B-cellmarkers). CD20 is a human B cell marker that is expressed during earlypre-B cell development and remains until plasma cell differentiation.The CD20 molecule may regulate a step in the activation process that isrequired for cell cycle initiation and differentiation, and is usuallyexpressed at very high levels on neoplastic B cells. Thus, the CD20surface antigen can be targeted for treating B cell lymphomas. U.S. Pat.No. 5,736,137, herein incorporated by reference, describes the chimericantibody “C2B8” that binds the CD20 antigen and its use to treat B-celllymphoma (antibody is also known as Rituxan®, rituximab, Mabthera (thisis a trademark in Europe)).

In an embodiment, the antibody is a monoclonal antibody. Monoclonalantibodies are highly specific, being directed against a singleantigenic site, and each monoclonal antibody is directed against asingle determinant on the antigen. For example, the monoclonalantibodies to be used in accordance with the present disclosure may bemade by the hybridoma method first described by Kohler et al., 1975,Nature 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described in Clacksonet al., 1991, Nature 352:624-628, and Marks et al., 1991, J. Mol. Biol.222:581-597, for example. For clinical use, the monoclonal antibodiesmay be humanized forms of non-human antibodies. These are chimericantibodies that contain sequences derived from both human and non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins in which residues from a hypervariable region of therecipient are replaced by residues from a hypervariable region of anon-human species having the desired specificity, affinity, andcapacity.

Specificity, as used herein, refers to the affinity of the antibody, andto the cross-reactivity with other antigens. In order to consider anantibody interaction to be “specific”, the affinity will be at leastabout 10⁻⁷ M, usually about 10⁻⁸ to 10⁻⁹ M, and may be up to 10⁻¹¹ M orhigher for the epitope of interest. It will be understood by those ofskill in the art that the term “specificity” refers to such a highaffinity binding, and is not intended to mean that the antibody cannotbind to other molecules as well. One may find cross-reactivity withdifferent epitopes, due, e.g., to a relatedness of antigen sequence orstructure, or to the structure of the antibody binding pocket itself.Antibodies demonstrating such cross-reactivity are still consideredspecific for the purposes of the present disclosure.

In practicing the subject methods, a subject or patient sample, e.g.,cells or collections thereof, e.g., tissues, is assayed to determinewhether the host or subject from which the assayed sample was obtainedis responsive to a given therapy, e.g., therapeutic antibody therapy.The sample obtained from the host is assayed to determine the genotypeof the host or subject from which the sample was obtained with respectto at least two or more, polymorphisms, where polymorphisms of interestare referred to herein as target polymorphisms.

In certain embodiments, the target polymorphisms are FcγR polymorphisms.An FcγR polymorphism is a polymorphism present in an FcγR (Fc receptor)protein. FcγR proteins of interest include, but are not limited to,FcγRII proteins (e.g., FcγRIIA, also known as CD32 (whose amino acid andnucleotide sequence is present at Genbank accession NOs. NM_(—)021642 orM28697)); FcγRIII proteins (e.g., FcγRIIIA, also known as CD16 (whoseamino acid and nucleotide sequence is present at Genbank accession NOs.BCO36723; BCO33678; BC017865 and NM_(—)000569)).

In certain embodiments, the sample is assayed to determine the genotypeof a subject with respect to two or more different target polymorphisms,where the two or more different target polymorphisms include at leastone FcγR polymorphism. In certain of these embodiments, at least two ofthe target polymorphisms are different FcγR polymorphisms, such as anFcγRII and an FcγRIII polymorphism.

In some embodiments, the sample is assayed to determine the genotype ofthe subject with respect to at least two or more target polymorphisms,where, the target polymorphisms are an FcγRII polymorphism and anFcγRIII polymorphism. In certain embodiments, the FcγRIIA polymorphismis the FcγRIIA H/R¹³¹ polymorphism (where the nucleotide codons encodingthe H and R residues of the polymorphism are CAT and CGT, respectively).In certain embodiments, the FcγRIIIA polymorphism is the FcγRIIIA V/F¹⁵⁸polymorphism (where the nucleotide codons encoding the V and F residuesof the polymorphism are GTT and TTT, respectively).

In practicing the subject methods, a subject or patient sample, e.g.,cells or collections thereof, e.g., tissues, is assayed to determinewhether the host or subject from which the assayed sample was obtainedis responsive to a given therapy, e.g., therapeutic antibody therapy. Inpracticing the subject diagnostic methods, the sample obtained from thehost is assayed to determine the genotype of the host or subject fromwhich the sample was obtained with respect to at least one, i.e., one ormore, polymorphisms, where polymorphisms of interest are referred toherein as target polymorphisms. In certain embodiments, the at least onetarget polymorphism is an FcγR polymorphism. An FcγR polymorphism is apolymorphism present in an FcγR (Fc receptor) protein. FcγR proteins ofinterest include, but are not limited to, FcγRII proteins (e.g.,FcγRIIA, also known as CD32 (whose amino acid and nucleotide sequence ispresent at Genbank accession nos. NM_(—)021642 or M28697)); FcγRIIIproteins (e.g., FcγRIIIA, also known as CD16 (whose amino acid andnucleotide sequence is present at Genbank accession nos. BCO36723;BCO33678; BC017865 and NM 000569)), and the like. In certainembodiments, the sample is assayed to determine the genotype of the hostwith respect to a single target polymorphism, where in theseembodiments, the single target polymorphism is an FcγRII polymorphism,such as an FcγRIIA polymorphism, where a specific representative FcγRIIApolymorphism of interest is the FcγRIIA H/R¹³¹ polymorphism (where thenucleotide codons encoding the H and R residues of the polymorphism areCAT and CGT, respectively). In certain embodiments, the sample isassayed to determine the genotype of the host with respect to two ormore different target polymorphisms, where in these embodiments, the twoor more different target polymorphisms include at least one FcγRpolymorphism. In certain of these embodiments, at least two of thetarget polymorphisms are different FcγR polymorphisms, such as an FcγRIIand an FcγRIII polymorphism. In certain embodiments, the sample isassayed for both an FcγRII polymorphism, such as the specific FcγRIIApolymorphisms described above, and an FcγRIII polymorphism, such as anFcγRIIIA polymorphism, including the FcγRIIIA V/F¹⁵⁸ polymorphism (wherethe nucleotide codons encoding the V and F residues of the polymorphismare GTT and TTT, respectively).

Any convenient protocol for assaying a sample for the above one or moretarget polymorphisms may be employed in the subject methods. In certainembodiments, the target polymorphism will be detected at the proteinlevel, e.g., by assaying for a polymorphic protein. In yet otherembodiments, the target polymorphism will be detected at the nucleicacid level, e.g., by assaying for the presence of nucleic acidpolymorphism, e.g., a single nucleotide polymorphism (SNP) that causeexpression of the polymorphic protein.

For example, polynucleotide samples derived from (e.g., obtained from)an individual may be employed. Any biological sample that comprises apolynucleotide from the individual is suitable for use in the methods ofthe disclosure. The biological sample may be processed so as to isolatethe polynucleotide. Alternatively, whole cells or other biologicalsamples may be used without isolation of the polynucleotides containedtherein. Detection of a target polymorphism in a polynucleotide samplederived from an individual can be accomplished by any means known in theart, including, but not limited to, amplification of a sequence withspecific primers; determination of the nucleotide sequence of thepolynucleotide sample; hybridization analysis; single strandconformational polymorphism analysis; denaturing gradient gelelectrophoresis; mismatch cleavage detection; and the like. Detection ofa target polymorphism can also be accomplished by detecting analteration in the level of a mRNA transcript of the gene; aberrantmodification of the corresponding gene, e.g., an aberrant methylationpattern; the presence of a non-wild-type splicing pattern of thecorresponding mRNA; an alteration in the level of the correspondingpolypeptide; and/or an alteration in corresponding polypeptide activity.

Detection of a target polymorphism by analyzing a polynucleotide samplecan be conducted in a number of ways. A test nucleic acid sample can beamplified with primers which amplify a region known to comprise thetarget polymorphism(s). Genomic DNA or mRNA can be used directly.Alternatively, the region of interest can be cloned into a suitablevector and grown in sufficient quantity for analysis. The nucleic acidmay be amplified by conventional techniques, such as a polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in a variety of publications,including, e.g., “PCR Protocols (Methods in Molecular Biology)” J. M. S.Bartlett and D. Stirling, eds, Humana Press (2000); and “PCRApplications: Protocols for Functional Genomics” Innis, Gelfand, andSninsky, eds., Academic Press (1999). Once the region comprising atarget polymorphism has been amplified, the target polymorphism can bedetected in the PCR product by nucleotide sequencing, by SSCP analysis,or any other method known in the art. In performing SSCP analysis, thePCR product may be digested with a restriction endonuclease thatrecognizes a sequence within the PCR product generated by using as atemplate a reference sequence, but does not recognize a correspondingPCR product generated by using as a template a variant sequence byvirtue of the fact that the variant sequence no longer contains arecognition site for the restriction endonuclease. PCR may also be usedto determine whether a polymorphism is present by using a primer that isspecific for the polymorphism. Such methods may comprise the steps ofcollecting from an individual a biological sample comprising theindividual's genetic material as template, optionally isolating templatenucleic acid (genomic DNA, mRNA, or both) from the biological sample,contacting the template nucleic acid sample with one or more primersthat specifically hybridize with a target polymorphic nucleic acidmolecule under conditions such that hybridization and amplification ofthe template nucleic acid molecules in the sample occurs, and detectingthe presence, absence, and/or relative amount of an amplificationproduct and comparing the length to a control sample. Observation of anamplification product of the expected size is an indication that thetarget polymorphism contained within the target polymorphic primer ispresent in the test nucleic acid sample. Parameters such ashybridization conditions, polymorphic primer length, and position of thepolymorphism within the polymorphic primer may be chosen such thathybridization will not occur unless a polymorphism present in theprimer(s) is also present in the sample nucleic acid. Those of ordinaryskill in the art are well aware of how to select and vary suchparameters. See, e.g., Saiki et al., 1986, Nature 324:163; and Saiki etal, 1989, Proc. Natl. Acad. Sci. USA 86:6230. As one non-limitingexample, a PCR primer comprising the T78C variation described in Example1 may be used.

Alternatively, various methods are known in the art that utilizeoligonucleotide ligation as a means of detecting polymorphisms. See,e.g., Riley et al., 1990, Nucleic Acids Res. 18:2887-2890; and Delahuntyet al., 1996, Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g., fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g., ³²P, ³⁵5, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g., avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid may be sequenced by a dideoxy chain terminationmethod or other well-known methods. Genomic DNA or mRNA may be useddirectly. If mRNA is used, a cDNA copy may first be made. If desired,the sample nucleic acid can be amplified using a PCR. A variety ofsequencing reactions known in the art can be used to directly sequencethe relevant gene, or a portion thereof in which a specific polymorphismis known to occur, and detect polymorphisms by comparing the sequence ofthe sample nucleic acid with a reference polynucleotide that contains atarget polymorphism. Any of a variety of automated sequencing procedurescan be used. See, e.g., WO 94/16101; Cohen et al., 1996, Adv.Chromatography 36:127-162.

Hybridization with the variant sequence may also be used to determinethe presence of a target polymorphism. Hybridization analysis can becarried out in a number of different ways, including, but not limited toSouthern blots, Northern blots, dot blots, microarrays, etc. Thehybridization pattern of a control and variant sequence to an array ofoligonucleotide probes immobilized on a solid support, as described inU.S. Pat. No. 5,445,934, or in WO 95/35505, may also be used as a meansof detecting the presence of variant sequences. Identification of apolymorphism in a nucleic acid sample can be performed by hybridizing asample and control nucleic acids to high density arrays containinghundreds or thousands of oligonucleotide probes. Cronin et al., 1996,Human Mutation 7:244-255; and Kozal et al., 1996, Nature Med. 2:753-759.

Single strand conformational polymorphism (SSCP) analysis; denaturinggradient gel electrophoresis (DGGE); mismatch cleavage detection; andheteroduplex analysis in gel matrices can also be used to detectpolymorphisms. Alternatively, where a polymorphism creates or destroys arecognition site for a restriction endonuclease (restriction fragmentlength polymorphism, RFLP), the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels. Theaforementioned techniques are well known in the art. Detaileddescription of these techniques can be found in a variety ofpublications, including, e.g., “Laboratory Methods for the Detection ofMutations and Polymorphisms in DNA” G. R. Taylor, ed., CRC Press (1997),and references cited therein.

A number of methods are available for determining the expression levelof a polymorphic nucleic acid molecule, e.g., a polymorphic mRNA, orpolymorphic polypeptide in a particular sample. Diagnosis may beperformed by a number of methods to determine the absence or presence oraltered amounts of normal or abnormal mRNA in a patient sample. Forexample, detection may utilize staining of cells or histologicalsections with labeled antibodies, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g., fluorescein, rhodamine, Texas red, etc.Final detection uses a substrate that undergoes a color change in thepresence of the peroxidase. The absence or presence of antibody bindingmay be determined by various methods, including flow cytometry ofdissociated cells, microscopy, radiography, scintillation counting, etc.The presence and/or the level of a polymorphic polypeptide may also bedetected and/or quantitated in any of a variety of published procedures.

Alternatively, one may focus on the expression of mRNA. Biochemicalstudies may be performed to determine whether a sequence polymorphism ina coding region or control regions is associated with disease. Diseaseassociated polymorphisms may include deletion or truncation of the gene,mutations that alter expression level, that affect the activity of theprotein, etc.

Screening for mutations in a polymorphic polypeptide may be based on thefunctional or antigenic characteristics of the protein. Proteintruncation assays are useful in detecting deletions that may affect thebiological activity of the protein. Various immunoassays designed todetect polymorphisms in polymorphic polypeptides may be used inscreening. Where many diverse genetic mutations lead to a particulardisease phenotype, functional protein assays have proven to be effectivescreening tools. The activity of the encoded a polymorphic polypeptidemay be determined by comparison with a reference polypeptide lacking aspecific polymorphism.

Diagnostic methods of the subject disclosure in which the level ofpolymorphic gene expression is of interest will typically involvecomparison of the relevant nucleic acid abundance of a sample ofinterest with that of a control value to determine any relativedifferences, where the difference may be measured qualitatively and/orquantitatively, which differences are then related to the presence orabsence of an abnormal gene expression pattern. A variety of differentmethods for determining the nucleic acid abundance in a sample are knownto those of skill in the art, where particular methods of interestinclude those described in: Pietu et al., 1996, Genome Res. 6:492-503;Zhao et al., Gene (Apr. 24, 1995) 156: 207-213; Soares, 1997, Curr.Opin. Biotechnol. 8: 542-546; Raval, J., 1994, Pharmacol. Toxicol.Methods 32:125-127; Chalifour et al., 1994, Anal. Biochem. 216:299-304;Stolz & Tuan, 1996, Mol. Biotechnol. 6:225-230; Hong et al., 1982,Bioscience Reports 2: 907; and McGraw, 1984, Anal. Biochem. 143: 298.Also of interest are the methods disclosed in WO 97/27317, thedisclosure of which is herein incorporated by reference.

Additional references describing various protocols for detecting thepresence of a target polymorphism include, but are not limited to, thosedescribed in: U.S. Pat. Nos. 6,703,228; 6,692,909; 6,670,464; 6,660,476;6,653,079; 6,632,606; and 6,573,049; the disclosures of which are hereinincorporated by reference.

Following obtainment of the genotype from the sample being assayed, thegenotype is evaluated to determine whether the subject is responsive tothe antineoplastic therapy of interest. In certain embodiments, theobtained genotype may be compared with a reference or control to make adiagnosis regarding the therapy responsive phenotype of the cell ortissue, and therefore host, from which the sample was obtained/derived.The terms “reference” and “control” as used herein mean a standardizedgenotype to be used to interpret the genotype of a given patient andassign a prognostic class thereto. The reference or control may be agenotype that is obtained from a cell/tissue known to have the desiredphenotype, e.g., responsive phenotype, and therefore may be a positivereference or control genotype. In addition, the reference/controlgenotype may be from a cell/tissue known to not have the desiredphenotype, and therefore be a negative reference/control genotype.

In certain embodiments, the obtained genotype is compared to a singlereference/control genotype to obtain information regarding the phenotypeof the cell/tissue being assayed. In yet other embodiments, the obtainedgenotype is compared to two or more different reference/control profilesto obtain more in depth information regarding the phenotype of theassayed cell/tissue. For example, the obtained genotype may be comparedto a positive and negative genotype to obtain confirmed informationregarding whether the cell/tissue has the phenotype of interest.

Representative examples of genotypes associated with therapyresponsiveness, particularly Rituximab responsive include, but are notlimited to: the FcγRIIA H/H¹³¹ genotype and the FcγRIIIA V/V¹⁵⁸genotype. Representative examples of genotypes associated with therapynon-responsiveness, particularly Rituximab-non responsiveness include,but are not limited to: the FcγRIIA H/R¹³¹ genotype; the FcγRIIA R/R¹³¹genotype; the FcγRIIIA V/F¹⁵⁸ genotype; and the FcγRIIIA F/F¹⁵⁸genotype.

In certain embodiments, the above-obtained information about thecell/tissue being assayed is employed to diagnose a host, subject orpatient with respect to responsive to therapeutic antibody therapy, asdescribed above. In certain embodiments, the above-obtained informationis employed to give a refined probability determination as to whether asubject will or will not respond to a particular therapy. For example,an identification of the FcγRIIA H/H¹³¹ genotype and/or the FcγRIIIAV/V¹⁵⁸ genotype may be employed to determine that the subject has atleast a 70% chance, such as at least a 75% chance, including at least an80% chance of responding to antibody, e.g., Rituximab, therapy.Likewise, an identification of the FcγRIIA H/R¹³¹ or R/R¹³¹ genotypeand/or the FcγRIIIA V/F¹⁵⁸ or F/F¹⁵⁸ genotype may be employed todetermine that the subject has less than 50% chance, such as a less than45% chance, including a less than 40% chance of responding to antibody,e.g., Rituximab, therapy.

The subject methods further find use in pharmacogenomic applications. Inthese applications, a subject is first diagnosed for the presence ofabsence of a responsive phenotype using a protocol such as thediagnostic protocol described in the preceding section.

The subject is then treated using a pharmacological protocol, where thesuitability of the protocol for a particular subject/patient isdetermined using the results of the diagnosis step. More specifically,where the identified phenotype is responsive, an appropriate therapeuticantibody treatment protocol is then employed to treat the patient.Alternatively, where a patient is identified as having a non-responsivephenotype, other treatment options are sought.

Methods for Selecting a Patient for Treatment with an Antibody

Methods are provided for selecting a patient for treatment with anantibody or antibody fragment based on the patient's FcγRIIA andFcγRIIIA polymorphism. For example, a patient with a particular FcγRIIA(H/R¹³¹) polymorphism and an FcγRIIIA (V/F¹⁵⁸) polymorphism is selectedfor treatment with one or more antibody therapies.

Methods are provided for selecting a patient for treatment with anantibody, comprising: (a) determining if the patient has an FcγRIIIAV/V¹⁵⁸ genotype, an FcγRIIIA V/F¹⁵⁸ or an FcγRIIIA F/F¹⁵⁸ genotype; (b)determining if the patient has an FcγRIIA H/H¹³¹ genotype, an FcγRIIAH/R¹³¹ genotype or an FcγRIIA R/R¹³¹ genotype; (c) selecting the patientwith a particular genotype for treatment with the antibody based on thegenotype determination of steps (a) and (b); and (d) administering theantibody to the patient selected in step (c).

A patient is assigned to one of nine Groups according to theirFcγRIIA(H/R¹³¹) polymorphism and an FcγRIIIA (V/F¹⁵⁸) polymorphism.Genotypes include: V/V¹⁵⁸, H/H¹³¹ (Group-I); V/F¹⁵⁸, H/H¹³¹ (Group-II);F/F¹⁵⁸, H/H¹³¹ (Group-III); V/V¹⁵⁸, H/R¹³¹ (Group-IV); V/F¹⁵⁸, H/R¹³¹(Group-V); F/F¹⁵⁸, H/R¹³¹ (Group-VI); V/V¹⁵⁸, R/R¹³¹ (Group-VII);V/F¹⁵⁸, R/R¹³¹ (Group-VIII); and F/F¹⁵⁸, R/R¹³¹ (Group-IX). Accordingly,a patient with one of the above-described genotypes is selected fortreatment with one or more antibody therapies.

In some embodiments, the antibody, variable region of an antibody or Fcvariant antibody is selected from the Group RITUXAN®, CAMPATH®,ZENAPAX®, HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®, REMICADE®, HUMIRA®,ERBITUX®, SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®, MYLOTARG®, REOPRO®,LUCENTIS®, SOURIS®, OKT3®, BEXXAR® ZEVALIN®, ENBREL® or AMEVIVE®.

Methods of Determining a Degree of Responsiveness

Methods are provided for determining the degree of treatment responsethat an individual having an ADCC-treatable disease or disorder willhave to an antibody therapy for the disease or disorder. The methodsgenerally involve: a) genotyping an individual having a disease ordisorder that is treatable with an ADCC-based antibody therapy, todetermine an FcγRIIA polymorphism to obtain a first result; b)genotyping the individual to determine an FcγRIIIA polymorphism toobtain a second result; and c) employing the first and second results todetermine the degree of responsiveness of the individual to the antibodytherapy.

Diseases and disorders that are treatable with an ADCC-based antibodytherapy include, but are not limited to, neoplastic diseases; autoimmunediseases; allograft rejection, and microbial infections.

Thus, e.g., in some embodiments, methods are provided for determiningthe degree of responsiveness that an individual having a neoplasticdisease will have to an antibody therapy. The methods generally involve:a) genotyping an individual having a neoplastic disease, to determine anFcγRIIA polymorphism to obtain a first result; b) genotyping theindividual to determine an FcγRIIIA polymorphism to obtain a secondresult; and c) employing the first and second results to determine thedegree of responsiveness of the individual to the antibody therapy.

In other embodiments, methods are provided for determining the degree ofresponsiveness that a subject having an ADCC-treatable disease ordisorder will have to an antibody therapy for the disease or disorder,the method comprising: (a) genotyping the subject for an FcγRIIApolymorphism and an FcγRIIIA polymorphism; and (b) identifying agenotype associated with a particular degree of responsiveness to theantibody therapy from a reference that is identical to the genotype fromthe test subject, wherein the test subject is determined to have adegree of responsiveness to the antibody therapy for the disease ordisorder corresponding to the level of responsiveness associated withthe reference having an identical FcγRIIA polymorphism and an identicalFcγRIIIA polymorphism.

In a further embodiment, methods are provided for determining the degreeof responsiveness that a test subject having an ADCC-treatable diseaseor disorder will have to an antibody therapy for the disease ordisorder, the method comprising: (a) genotyping subjects for an FcγRIIApolymorphism and an FcγRIIIA polymorphism, wherein the subjects have orhad the ADCC-treatable disease or disorder and are or were administeredantibody therapy for the disease or disorder; (b) classifying eachsubject based on its FcγRIIA polymorphism and FcγRIIIA polymorphism toone of more than three categories of responsiveness to the antibodytherapy; (c) genotyping the test subject for an FcγRIIA polymorphism andan FcγRIIIA polymorphism; and (d) identifying a genotype from (a) thatis identical to the genotype from the test subject in step (c), whereinthe test subject is determined to have a degree of responsiveness to theantibody therapy for the disease or disorder corresponding to the levelof responsiveness associated with a subject having an identical FcγRIIApolymorphism and an identical FcγRIIIA polymorphism.

The predicted responsiveness of an individual to an antibody therapy maybe categorized into a plurality of different categories based on firstand second results, such as three or more different categories, e.g., a)“high degree of responsiveness”: the individual has at least a 65%chance, at least a 70% chance, at least a 75% chance, or at least an 80%chance, or greater, of responding to the antibody therapy; b)“intermediate degree of responsiveness”: the individual has from about a35% chance to about a 65% chance, or from about a 50% chance to about a70% chance, of responding to the antibody therapy; and c) “low degree ofresponsiveness”: the individual has a less than 50% chance, less than45% chance, or less than 40% chance, of responding to the antibodytherapy.

Responsiveness may be determined at various times after treatment with agiven antibody therapy, e.g., 1-3 months, 3-6 months, 6-9 months, or9-12 months following treatment, e.g., following initiation oftreatment. Thus, e.g., responsiveness can be expressed with a timecomponent.

For example, in some embodiments, predicted responsiveness of anindividual to an antibody therapy may be categorized into a plurality ofdifferent categories based on first and second results, such as three ormore different categories, e.g., a) “high degree of responsiveness”: theindividual has at least a 65% chance, at least a 70% chance, at least a75% chance, or at least an 80% chance, or greater, of responding to theantibody therapy at one month following initiation of treatment; b)“intermediate degree of responsiveness”: the individual has from about a35% chance to about a 65% chance, or from about a 50% chance to about a70% chance, of responding to the antibody therapy at one month followinginitiation of treatment; and c) “low degree of responsiveness”: theindividual has a less than 50% chance, less than 45% chance, or lessthan 40% chance, of responding to the antibody therapy at one monthfollowing initiation of treatment.

In other embodiments, predicted responsiveness of an individual to anantibody therapy may be categorized into a plurality of differentcategories based on first and second results, such as three or moredifferent categories, e.g., a) “high degree of responsiveness”: theindividual has at least a 65% chance, at least a 70% chance, at least a75% chance, or at least an 80% chance, or greater, of responding to theantibody therapy at three months following initiation of treatment; b)“intermediate degree of responsiveness”: the individual has from about a35% chance to about a 65% chance, or from about a 50% chance to about a70% chance of responding to the antibody therapy at three monthsfollowing initiation of treatment; and c) “low degree ofresponsiveness”: the individual has a less than 50% chance, less than45% chance, or less than 40% chance, of responding to the antibodytherapy at three months following initiation of treatment.

In other embodiments, predicted responsiveness of an individual to anantibody therapy may be categorized into a plurality of differentcategories based on first and second results, such as three or moredifferent categories, e.g., a) “high degree of responsiveness”: theindividual has at least a 65% chance, at least a 70% chance, at least a75% chance, or at least an 80% chance, or greater, of responding to theantibody therapy at six months following initiation of treatment; b)“intermediate degree of responsiveness”: the individual has from about a35% chance to about a 65% chance, or from about a 50% chance to about a70% chance of responding to the antibody therapy at six months followinginitiation of treatment; and c) “low degree of responsiveness”: theindividual has a less than 50% chance, less than 45% chance, or lessthan 40% chance, of responding to the antibody therapy at six monthsfollowing initiation of treatment.

In other embodiments, predicted responsiveness of an individual to anantibody therapy may be categorized into a plurality of differentcategories based on first and second results, such as three or moredifferent categories, e.g., a) “high degree of responsiveness”: theindividual has at least a 65% chance, at least a 70% chance, at least a75% chance, or at least an 80% chance, or greater, of responding to, theantibody therapy at 12 months following initiation of treatment; b)“intermediate degree of responsiveness”: the individual has from about a35% chance to about a 65% chance, or from about a 50% chance to about a70% chance of responding to the antibody therapy at 12 months followinginitiation of treatment; and c) “low degree of responsiveness”: theindividual has a less than 50% chance, less than 45% chance, or lessthan 40% chance, of responding to the antibody therapy at 12 monthsfollowing initiation of treatment.

Responsiveness to an antibody therapy for a neoplastic disease caninclude one or more of: antibody-dependent cell-mediated cytotoxicity(ADCC) response to tumor cells; reduction in tumor mass; reduction innumber of tumor cells; etc. Responsiveness to an antibody therapy for anautoimmune disease can include one or more of: reduction in a symptomassociated with the autoimmune disorder; reduction in the number and/oractivity of an autoreactive B-cell; reduction in the number and/oractivity of an autoreactive T-cell; etc. Responsiveness to an antibodytherapy for allograft rejection can include one or more of: reduction inthe amount of immunosuppressive drug that must be administered to anindividual who is the recipient of an allograft and still maintain theallograft; duration of maintenance of the allograft; function of theallograft; reduction in the number and/or activity of alloreactiveT-cells in the allograft recipient. Responsiveness to an antibodytherapy for a viral infection can include one or more of: reduction inthe number of viral genomes in a tissue, fluid, or other specimen froman individual; reduction in one or more symptoms of a viral infection;etc. Responsiveness can also be assessed using an in vitro ADCC assay,e.g., as described in the Examples.

For example, where an FcγRIIIA polymorphism can be one of A/A, A/a, anda/a, and where an FcγRIIA polymorphism can be one of B/B, B/b, and b/b,responsiveness to a given antibody therapy at a given time point aftertreatment may be categorized as shown in Tables A and B, below. A/A,a/a, B/B, and b/b represent homozygous alleles for the respective FcγRpolymorphisms, while A/a and B/b represent heterozygous alleles for therespective FcγR polymorphisms.

TABLE A FcγRIIIA FcγRIIIA FcγRIIIA Genotype A/A Genotype A/a Genotypea/a Fcγ RIIA  70%-100% 50%-70% 50%-70% Genotype B/B Fcγ RIIA 50%-70%<50% <50% Genotype B/b Fcγ RIIA 50%-70% <50% <50% Genotype b/b

TABLE B Allelic expression patterns based on FcγRIIIA and FcγRIIApolymorphisms in various patient Groups Fcγ RIIIA Fcγ RIIIA Fcγ RIIIAGenotype A/A Genotype A/a Genotype a/a Fcγ RIIA Group I Group II GroupIII Genotype B/B 70%-100% 50%-70% 50%-70% A, A; B, B A, a; B, B a, a; B,B Fcγ RIIA Group IV Group V <50% Group VI <50% Genotype B/b 50%-70% A,a; B, b a, a; B, b A, A; B, b Fcγ RIIA Group VII Group VIII <50% GroupIX <50% Genotype b/b 50%-70% A, a; b, b a, a; b, b A, A; b, b

For a given antibody (a “reference antibody”) with responsivenesscategories as set forth in Table A or Table B, Table A or Table B is anexemplary reference chart. A person with unknown responsiveness totherapy with the reference antibody is genotyped for FcγRIIA andFcγRIIIA polymorphisms; and the person's genotype is compared with thereference chart; and the degree of predicted responsiveness isdetermined from the reference chart. In some embodiments, where thepredicted degree of responsiveness is high, the individual will betreated with an antibody that is the same as the reference antibody. Inother embodiments, where the predicted degree of responsiveness isintermediate (e.g., 50%-70%) or low (e.g., <50%), a therapeutic antibodywill be selected that has the same antigen binding specificity as thereference antibody, but has enhanced in vitro ADCC activity to anFcγRIIA and/or an FcγRIIIA, as described in more detail below. Thenature of allelic expression for the above genotypes can be bestdescribed by codominance. For example, for the Patient Group-V (Table B)with genotypes FcγRIIIA (A/a) and FcγRIIA (B/b) will have equalphenotypic expression of both alleles A,a for FcγRIIIA and B,b forFcγRIIA. In some embodiments, individuals of category (a) will have anFcγRIIA H/H¹³¹ genotype and an FcγRIIIA V/V¹⁵⁸ genotype. In someembodiments, individuals of category (b) will have an FcγRIIA H/H¹³¹genotype or an FcγRIIIA V/V¹⁵⁸ genotype, but not both. For example,individuals of category (b) will have one of: i) an FcγRIIA H/H¹³¹genotype and an FcγRIIIA V/F¹⁵⁸ genotype; ii) an FcγRIIA H/H¹³¹ genotypeand an FcγRIIIA 158 F/F genotype; iii) an FcγRIIA H/R¹³¹ genotype and anFcγRIIIA V/V¹⁵⁸ genotype; or iv) an FcγRIIA R/R¹³¹ genotype and anFcγRIIIA V/V¹⁵⁸ genotype. Individuals of category (c) will have neitheran FcγRIIA H/H¹³¹ genotype nor an FcγRIIIA V/V¹⁵⁸ genotype. Thus, e.g.,in some embodiments, individuals of category (c) will have one of: i) anFcγRIIA H/R¹³¹ genotype and an FcγRIIIA V/F¹⁵⁸ genotype; ii) an FcγRIIAR/R¹³¹ genotype and an FcγRIIIA V/F¹⁵⁸ genotype; iii) an FcγRIIA H/R¹³¹genotype and an FcγRIIIA F/F¹⁵⁸ genotype; or iv) an FcγRIIA R/R¹³¹genotype and an FcγRIIIA F/F¹⁵⁸ genotype. The nature of allelicexpression for the above genotypes can be best described by codominance.For example, for the Patient Group-V (Table B) with genotypes FcγRIIIA(V/F) and FcγRIIA (H/R) will have equal phenotypic expression of bothalleles V,F for FcγRIIIA and H,R for FcγRIIA. Both FcγRIIIA and FcγRIIAare monomers under physiological conditions, and the number of thesereceptors can be .about.15,000-35,000 per cell (Guyre et al., 1983, J.Clin. Invest. 72:393-397). Individuals with an FcγRIIIA V/F¹⁵⁸ genotypewill express equal number of V¹⁵⁸ and F¹⁵⁸ alleles. Similarly,individuals with an FcγRIIA 131H/R genotype will express equal number ofH¹³¹ and R¹³¹ alleles. As an example, a reference chart with variouscombinations of FcγRIIA and FcγRIIIA polymorphisms, and correspondingcategories of anticipated responsiveness to a monoclonal antibodytherapy (e.g., Rituxan), are shown in Table C, below.

TABLE C FcγRIIIA 158 V/V FcγRIIIA V IF FcγRIIIA F/F FcγRIIA 131 H/H 70%-100% 50%-70% 50%-70% FcγRIIA 131 H/R 50%-70% <50% <50% FcγRIIA 131R/R 50%-70% <50% <50%

TABLE D Allelic expression patterns based on FcγRIIIA and FcγRIIApolymorphisms in various patient Groups (n = 87) FcγRIIIA V/V¹⁵⁸FcγRIIIA V/F¹⁵⁸ FcγRIIIA F/F¹⁵⁸ Total (%) FcγRIIA Group-I 70%-100%Group-II 50%-70% Group-III 50%-70% 20 (23)  H/H¹³¹ V, V; H, H V, F; H, HF, F; H, H 3 (3.4) 14 (16.1) 3 (3.4) FcγRIIA Group-IV 50%-70% Group-V<50% Group-VI <50% 19 (21.8) H/R¹³¹ V, V; H, R V, F; H, R F, F; H, R 8(9.2) 16 (18.4) 19 (21.8) FcγRIIA Group-VII 50%-70% Group-VIII <50%Group-IX <50% 12 (13.8) R/R¹³¹ V, V; R, R V, F; R, R F, F; R, R 2 (2.3)10 (11.8) 12 (13.8) Total (%) 13 (14.9) 40 (46) 34 (39.1) 87 (100) 

Table D is another example of a reference chart. The V¹⁵⁸ allele inFcγRIIIA is a high-affinity/high-responder receptor while the F¹⁵⁸allele is a low-affinity/low-responder receptor. Similarly, the H¹³¹allele in FcγRIIA is a high-affinity/high-responder receptor while theR¹³¹ allele is a low-affinity/low-responder receptor. The polymorphismswere determined in an expanded Group of 87 patients with follicularlymphoma (Table D; FIG. 13). The allelic frequencies are: pV=0.38,pF=0.62; pH=0.48; pR=0.52.

Further, FcγRIIIA and FcγRIIA were determined for populations of healthyU.S. Caucasians, healthy U.S. African Americans and healthy Norwegians.Interestingly, these groups exhibited a similar allelic expressionpattern as the patient group with B-NHL (FIG. 14).

As an example, a reference chart with various combinations of FcγRIIAand FcγRIIIA polymorphisms, and corresponding categories of anticipatedresponsiveness to a monoclonal antibody therapy (e.g., Rituxan), areshown in Table D, above.

In some embodiments, an FcγRIIIA genotype and an FcγRIIA genotype aredetermined using a nucleic acid probe that hybridizes under stringenthybridization conditions to FcγRIIA-encoding and FcγRIIIA-encodingnucleic acids comprising polymorphisms associated with an alteredresponse to treatment with a therapeutic antibody. FcγRIIA-encoding andFcγRIIIA-encoding nucleic acids (referred to collectively herein as“target nucleic acids”) are in some embodiments genomic DNA thatcomprise nucleotide sequences encoding all or part of an FcγRIIA or anFcγRIIIA, and that include a polymorphism associated with a response toan antibody therapeutic.

In other embodiments, an FcγRIIIA genotype and an FcγRIIA genotype aredetermined using nucleic acid primer pairs that, in the presence of anappropriate polymerase and other reagents (e.g., dNTPs, ions such asmagnesium ions, etc.), prime the synthesis of an amplification productusing the target nucleic acids as templates, using, e.g., a polymerasechain reaction. The target nucleic acids are chosen such that theyinclude at least one polymorphic region. For example, a first primerpair primes amplification of an FcγRIIA target nucleic acid thatcomprises the polymorphism that gives rise to H/H¹³¹, H/R¹³¹, or R/R¹³¹;and a second primer pair primes amplification of an FcγRIIIA targetnucleic acid that comprises a polymorphism that gives rise to V/V¹⁵⁸,V/F¹⁵⁸, or F/F¹⁵⁸. A nucleic acid primer will in some embodimentsinclude a detectable label, which detectable label is incorporated intothe amplification product, giving rise to a detectably labeledamplification product. A nucleic acid primer will in some embodimentsinclude a restriction endonuclease recognition site not found in thetemplate or in other nucleic acid primers, such that an amplificationproduct is generated which includes a restriction endonucleaserecognition site that provides for its identification.

ADCC Mediated by NK-Cells and Macrophages

The presence of infiltrating NK cells and macrophages in surgicallyremoved tumors from MAb-treated patients has been extensively documented(Adams et al., 1984, Proc. Natl. Acad. Sic. USA 81:3506; Shetye et al.,1988, Cancer Immunol. Immunother. 27:154). Although both ADCC and CDCmay play a role in tumor cell destruction in vivo, as substantiated byseveral in vitro studies, the main anti-tumor mechanism of therapeuticantibodies in vivo is considered to be ADCC (See, e.g., Velders et al.,1998, British J. Cancer 78:478).

ADCC can be classified into two types: NK-cell mediated ADCC (NK-ADCC)and macrophage-mediated ADCC (M-ADCC). Both mechanisms eitherindependently or together can be viewed to play significant roles inmediating cytotoxicities, and have direct relevance in determining theclinical efficacy of MAb therapies (Carton et al., 2002, Blood 99:754;Weng and Levy, 2003, J. Clin. Oncol. 21:3940; Cheung et al., 2006, J.Clin. Oncol. 24:2885).

Most tumor cells appear to secrete chemoattractants which activelyrecruit monocytes to tumor sites (Graves et al., 1989, Science 245:1490;Bottazzi et al., 1983, Science 220:210). The mechanism of ADCC by theseMDM (monocyte-derived macrophages) appears to involve phagocytosis ofintact tumor cells (Munn and Cheung, 1990, J. Exp. Med. 172:231; Munn etal., 1991, Cancer Research 51:1117). Thus, in one embodiment,macrophages are important in immunotherapeutic regimens involvinganti-tumor ADCC.

Macrophages express three FcγRs for IgG: the high-affinity receptor,FγRI, and the two low-affinity receptors, FcγRIIA and FcγRIIIA FcγRIavidly binds monomeric human IgG (K_(a).about.2×10⁸ M⁻¹), and thereforethe binding of monoclonal antibody is competitively inhibited by serumimmunoglobulin.

FcγRIIIA is present in NK cells (FcγRIIIA_(NK)) and macrophages(FcγRIIIA_(M)). FcγRIIA is preferentially expressed on macrophages andneutrophils, and not on lymphocytes. Although FcγRIIIA_(NK) andFcγRIIIA_(M) have identical protein cores, they each undergodifferential cell type-specific glycosylation: FcγRIIIA_(NK) isglycosylated with high mannose- and complex-type oligosaccharides, whileFcγRIIIA_(M) has no high mannose-type oligosaccharides. Because of this,FcγRIIIA_(NK) exhibits higher affinity for IgG₁ and this feature is notinfluenced by VF¹⁵⁸ allelic polymorphism (Edberg and Kimberly, 1997, J.Immunol. 159:3849). In normal whole blood or plasma (containing 8-11mg/ml IgG), FcγRIIIA_(NK) was fully blocked, but FcγRIIIA_(M) showedapproximately 60% blockade of the binding of mAB 3G8, which binds in ornear the ligand binding site. The ligand binding site of FcγRIIIA_(NK)was blocked with as little as 2 mg/ml of human IgG₁ while FcγRIIIA_(M)was only partially (30%) blocked at this concentration. In contrast,plasma containing approximately 26 mg/ml of IgG (obtained from ImmuneThrombocytopenic Purpura patients receiving therapeutic g-globulin)showed complete saturation of FcγRIIIA_(M) with blockade of 3G8 binding.Binding of mAB IV.3, an FcγRIIA-specific MAb, to the low affinityFcγRIIA on monocytes was unaltered by exposure of the cells to the sameITP patient plasmas. Thus, in one embodiment, the ligand bindingproperties of FcγRIIIA_(NK) and FcγRIIIA_(M) are distinctly different(Edberg and Kimberly, supra). In another embodiment, therefore, the ADCClevels mediated by the FcγRIIIA_(NK) and FcγRIIIA_(M) are expected to besignificantly different.

The FcγRIIIA VF¹⁵⁸ polymorphism is a clinically relevant phenotype thathas a direct impact on human biology (Wu et al., 1997, J. Clin. Invest.100:1059). Compared with FF¹⁵⁸ homozygotes, FcγRIIIA expressed on NKcells and monocytes in VV¹⁵⁸ homozygotes bound more IgG₁ and IgG₃despite identical levels of receptor expression. In response to astandard aggregated human IgG stimulus, FcγRIIIA engagement on NK cellsfrom VV¹⁵⁸ (high binding) homozygotes led to a larger rise in[Ca²⁺]_(i), a greater level of NK cell activation, and a more rapidinduction of activation-induced cell death (by apoptosis). Investigationof an independently phenotyped normal cohort revealed that all donorswith a low binding phenotype are FF¹⁵⁸ homozygotes, while all phenotypichigh binding donors have at least one V¹⁵⁸ allele (either VV or VF).Initial analyses of 200 patients with systemic lupus erythematosus (SLE)indicates a strong association of the low-binding phenotype (FF¹⁵⁸) withdisease, especially in patients with nephritis who have anunderrepresentation of the homozygous high binding (VV¹⁵⁸) phenotype(FF, 44%; VF, 46%; VV, 10%). This VF¹⁵⁸ polymorphism based variations inIgG binding was further correlated to the earlier observations that somepatients have “high” NK-cell mediated ADCC (Vance et al., 1993, J.Immunol. 151:6429).

Some forms of macrophage ADCC have been reported to be inhibited byserum IgG₁ which competes with monoclonal antibodies for binding toFcγRI. Hybridoma cells bearing surface antibody directed against eitherof FcγRs (II and III) were efficiently phagocytosed by MDM (Munn et.al., 1991, Cancer Research 51:1117). Soluble anti-receptor antibodiesagainst FcγRII and FcγRIII were able to inhibit ADCC but only when bothantibodies were simultaneously present suggesting that either FcγR iscapable of functioning independently to mediate phagocytosis of tumorcells. Consistent with the mechanism involving the low-affinityreceptors rather than FcγRI, antibody-dependent phagocytosis occurred inthe presence of human serum and purified human IgG. Greater than 75% ofthe MDM were able to ingest tumor cells when a suitable target cell wasavailable (Munn et. al., supra). Optimal phagocytosis occurred atmonoclonal antibody concentrations of 10-100 ng/ml. Because FcγRI isnormally occupied in vivo by serum IgG₁ the participation of bothlow-affinity FcγRs in tumor cell phagocytosis is potentially importantin establishing the in vivo applicability of this efficient form oftoxicity (Munn et. al., supra).

C-reactive protein (CRP) is involved in host defense, regulation ofinflammation, and modulation of autoimmune disease. CRP shares severalfunctional activities with IgG including binding to FcγRs (Crowell etal., 1991, J. Immunol. 147:3445; Marvell et al., 1995, J. Immunol.155:2185). Direct genetic evidence for FcγRIIA as the functional, highaffinity CRP receptor on monocytes/leukocytes has been provided, andthis study emphasized the reciprocal relationship between IgG and CRPavidities (Stein et. al., 2000, J. Clin. Invest. 105:369). FcγRIA bindsCRP with low affinity, whereas FcγRIIA binds CRP with high affinity. CRPbound with high avidity to monocytes and neutrophils from FcγRIIA-RR¹³¹homozygotes. CRP showed decreased binding to cells from FcγRIIA-HH¹³¹homozygotes. That is, both IgG₁, for instance, an antibody therapy suchas rituximab, and CRP will compete for the same binding site in FcγRIIA,and is further influenced by the HR¹³¹ polymorphism. FcγRIIA-HR¹³¹heterozygotes showed intermediate binding. These findings provide directgenetic evidence for FcγRIIA as the functional, high affinity CRPreceptor on leukocytes while emphasizing the reciprocal relationshipbetween IgG and CRP binding avidities (Stein et al. supra). Thus, in oneembodiment, the M-ADCC is the sum of ADCC mediated by the FcγRIIIA andFcγRIIA. In another embodiment, the M-ADCC is influenced by both VF¹⁵⁸and HR131 polymorphisms.

The murine IgG₃ anti-GD2 MAb 3F8 is being used in the clinic for itsantitumor activity in high-risk neuroblastoma patients. The MAb exhibitspotent in vitro ADCC activity (Kushner et. al., 1989, Blood 73:1936),and its phagocyte-mediated ADCC is markedly increased in the presence ofGM-CSF. ELISA studies showed 3F8 has preferential binding toFcγRIIA-R¹³¹ over FcγRIIA-H¹³¹. The role of FcγRIIA-HR¹³¹ andFcγRIIIA-VF¹⁵⁸ polymorphisms with clinical outcome of high-riskneuroblastoma patients (N=136) treated with 3F8 and GM-CSF wasinvestigated (Cheung et. al., 2006, J. Clin. Oncol. 24:2885).FcγRIIA-RR¹³¹ genotype correlated with progression-free survival for theentire cohort (P=0.049), and also correlated with marrow remission 2.5months after treatment initiation. This finding is in stark contrast towhat was shown for rituximab in B-NHL patients (Weng and Levy, supra),and can only be attributed to the fact that 3F8 is a mouse IgG₃ (Yan andDavis, 2006, Pharmacogenomics 7:961), whose hinge region is about fourtimes longer than that of IgG₁ (Michaelsen et. al., 1977, J. Biol. Chem.252:883, FIG. 18). Thus, in one embodiment, based on the H/R¹³¹polymorphisms, Fc variant IgG₃ antibodies can be generated with enhancedADCC activity.

Therefore, in some embodiments, the Fc engineering is focused onoptimizing both NK-ADCC and M-ADCC. In other embodiments, the Fcengineering is focused on optimizing the ADCC mediated by FcγRIIIA_(NK)and FcγRIIIA_(M). In some other embodiments, the Fc engineering isattempted to optimize the ADCC based on HR¹³¹ polymorphism in FcγRIIA.In other embodiments, the Fc engineering is attempted to optimize theADCC based on VF¹⁵⁸ polymorphism in FcγRIIIA.

Structure of Fc Region, Fc Receptors (FcR), and Fc-FcR Complexes

All known Fc receptors are members of the Ig super family, except forFccRII. The crystal structures of the extracellular domains of FcγRII(Maxwell et al., 1999, Nature Struct. Biol. 6:437-442; Sondermann etal., 1999, Biochemistry 29:8469-77) and FcγRIII (Zhang et al., 2000,Immunity 13:387-395) show remarkable similarities. The receptors consistof two extracellular Ig-like domains, D1 and D2, with acute interdomainhinge angles of 50-55°, unique to Fc receptors. The Fc-binding region islocated in the D2 domain. Additional crystal structures (Sondermann etal., 2000, Nature 406:267-73; Radaev et al., 2001, J. Biol. Chem.276:16469-77; Sondermann et al., 2001, J. Mol. Biol. 309:737-749) haverevealed that the receptor-ligand interface consists of the BC, C′E, FGloops and the Cβ. strand of the D2 domain, the hinge loop between the D1and D2 domains of the receptor providing additional interactions withFcγR (FIG. 12C).

In the complex structure, the receptor is asymmetrically bound betweenthe two Cγ2 domains of the Fc fragment creating a 1:1 receptor ligandstoichiometry. Only D2 of the sFcγRIII and two residues from the linkerconnecting this domain with D1 interact in the complex with differentregions of both Cγ2 domains (Cγ2-A and Cγ2-B) and the preceding hingeregions of hFc1 (Fc from human IgG1; Sondermann et al., 2000, Nature406:267-73; Sondermann et al., 2001, J. Mol. Biol. 309:737-749). BothFcγRIII and Fc components undergo substantial domain rearrangements uponbinding but are restricted to the loops involved in the complexformation.

Fc Region-sFcγRIII Contact Interface: sFcγRIII binds hFc1 with the B/Cloop (W¹¹⁰-A¹¹⁴) the F/G loop (V¹⁵⁵-K¹⁵⁸), the C strand (H¹¹⁶-T¹¹⁹) andC′ strand (D¹²⁶-H¹³²) on its carboxyl-terminal D2 (FIG. 12-C).Additionally, R¹⁵² and the connector between the N-terminal D1 and D2(I⁸⁵-W⁸⁷) is involved in binding. These regions interact with loops onthe hFc1 within Cγ2-B (B/C: D²⁶⁵-E²⁶⁹, C/E: N²⁹⁷-T²⁹⁹), with the F/Gloop of Cγ2A (A³²⁷-I³³²), with the carbohydrate residueN-acetyl-D-glucosamine (NAG)1 of Cγ2-B and with the hinge region of bothheavy chains (L²³⁴-S²³⁹). N²⁹⁷ is the glycosylation site in the Fcregion.

Two main contact areas exist. First, P³²⁹ of Cγ2-A is encaged tightly byW⁸⁷ and W¹¹⁰ of the FcγRIII (‘proline sandwich’). Second, residuesL²³⁴-S²³⁹ of the lower hinge of both hFc1 chains are engaged in sFcγRIIIbinding. These residues were disordered in the hFc1 crystal structuresbut are defined in the complex.

The receptor and the Fc fragment are in very close contact at the hingeregion. G²³⁷ of Cγ2-A and G²³⁶ of Cγ2-B adopt Phi/Psi angles in thecomplex not allowed for other residues. The hinge residues L²³⁴-S²³⁹ ofCγ2-A are in contact with residues T¹¹³, A¹¹⁴ and V¹⁵⁵-K¹⁵⁸ of FcγRIII.The main contact is mediated by L²³⁵ which binds in a shallowhydrophobic groove with V¹⁵⁵ at its bottom and the A¹¹⁴ of the B/C loop,and T¹¹³ Cγ2 at its rim (FIG. 12-C).

The hinge residues of the Cγ2-B domain are bound in a narrow channel ofsFcγRIII lined by H¹¹⁶ and H¹³² on one side and K¹¹⁷ on the other. Theresidues G²³⁶ and G²³⁷ of the hinge peptide bind into this channel andY¹²⁹ and H¹³¹ form additional contacts to the hinge peptide. In thisarrangement, the residues H¹¹⁶, H¹³¹, and H¹³² are potential hydrogenbond partners to hinge residues. L²³⁵ is in contact with H¹¹⁶ and H¹³²;this region is considerably more hydrophobic in other receptors.

The FcγRIII contacts the interstrand loops of the Cγ2-B domain and theterminal residues of the β-strands. The side chains of the residuesD¹²⁶-H¹³¹ (C′ strand) are bound to the residues D²⁶⁵-E²⁶⁹, and N²⁹⁷-T²⁹⁹of Cγ2-B. R¹⁵² of the F strand could potentially form a hydrogen bond tothe carbohydrate residue NAG1 that is directly attached to N²⁹⁷ ofCγ2-B. Some more distant contacts to this sugar residue are formed byK¹¹⁷, T¹¹⁹, D¹²⁶, and Y¹²⁹ (See, e.g., FIG. 12-C). Substitution ofAsparagine at position N²⁹⁷ should lead to aglycosylated Fc form, whichlacks ADCC activity but other functions such as CDC and FcRn binding areretained (Dorai et al., 1991, Hybridoma 10:211-217; Vaccaro et al.,2005, Nature Biotechnol. 23:1283-1287).

A sequence comparison of sFcγRIII with other FcγRs and the FccRIα showsa high degree of similarity (FIG. 12C). Mutagenesis studies with FcγRI(Canfield and Morrison, 1991, J. Exp. Med. 173:1483-91), FcγRII (Lund etal., 1991, J. Immunol. 147:2657-2662; Hulett et al., 1994, J. Biol.Chem. 269:15287-93; Hulett et al., 1995, J. Biol. Chem. 270:21188;Maxwell et al., 1999, Nature Struct. Biol. 6:437) and FcεRIα(Hulett etal., 1999, J. Biol. Chem. 274:13345) indicate that the same regions areinvolved in complex formation as are defined here for sFcγRIII.Therefore, based on several crystallographic and mutagenic studies, acommon model describing the principal interactions within the variouscomplexes can be proposed.

The proline sandwich appears as the primary binding motif in all FcR-Igcomplexes. The two tryptophan residues (W⁸⁷ and W¹¹⁰) are conserved inthe FcRs including FcεRIα and the proline occurs in all IgGs and IgE.Further conserved contacts are observed between K¹¹⁷ and D²⁶⁵, T¹¹⁹ andNAG1, and K¹²⁸ and E²⁶⁹ (the numbers in all Igs and FcRs refer to thehomologous position in IgG1 and sFcγRIII, respectively; FIG. 12C).

The hinge peptide is a second central recognition site. The sequencevariation in this region is likely to be the main reason for thediffering affinities in the FcR-Ig pairs. The interactions seem to bemodulated in different receptor Ig pairings in a productive ordestructive way by the non-conserved regions of the binding loops.

FcγRII and FcγRIII are 50% identical and the differences affect theloops in contact with the hinge, but not the contact regions to Cγ2-Aand Cγ2-B. The residues G²³⁶ and G²³⁷ of the Cγ2-B domain hinge peptideare bound in the narrow channel between V¹¹⁶, L¹³², L¹³² and K¹¹⁷. Theseexchanges at positions V¹¹⁶ and L¹³² create a small hydrophobic patch towhich L²³⁵ may bind and allow a tighter contact of receptor and hingepeptide.

Therefore, in some embodiments, amino acid sequence changes (e.g.,insertions, deletions, substitutions, etc.) in one or more of thefollowing conserved regions of IgG1-Fc region are expected to alter itsbinding properties to FcγRIIIA and FcγRIIA: L²³⁴LGGPS²³⁹; R²⁵⁵TPEVT²⁶⁰;D²⁶⁵VSHE²⁶⁹; N²⁹⁷ST²⁹⁹; A³²⁷LPAPI³³². In other embodiments, based on theV/F and H/R polymorphisms, Fc variant antibodies can be generated withenhanced binding, potency, efficacy, and ADCC function specific to thepatient Groups as exemplified in this disclosure. In some otherembodiments, modification of additional residues involved in Fcengineering is accomplished through Fc Walking.

Methods of Making Sets of Related Antibodies

Methods are provided for making a set of related antibodies. The methodgenerally involves:

a) modifying at least one Fc region amino acid residue in a parentantibody, such that the modified Fc region exhibits enhanced bindingaffinity to at least one Fc receptor encoded by an Fc receptor gene of afirst genotype, compared to the Fc binding affinity of the parentantibody, to generate a first Fc region modified antibody (also referredto as an “Fc variant antibody”); and

b) modifying at least one Fc region amino acid residue in a parentantibody, such that the modified Fc region exhibits enhanced bindingaffinity to at least one Fc receptor encoded by an Fc receptor gene of asecond genotype, compared to the Fc binding affinity of the parentantibody, to generate a second Fc region modified antibody.

In other embodiments, methods are provided to modify the Fc region of anantibody to reduce its binding affinity to at least one Fc receptorcompared to the Fc binding affinity of the parent antibody.

The parent antibody is a reference antibody. The first and second Fcregion modified antibodies each differ in Fc region amino acid sequenceby at least one amino acid from the Fc region amino acid sequence of theparent antibody, e.g., the first and the second Fc variant antibodieseach independently comprise an Fc region amino acid sequence thatdiffers in amino acid sequence from the Fc region of the parent antibodyby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more, aminoacids. In some embodiments, the first and the second Fc variantantibodies each independently comprise an Fc region amino acid sequencethat differs in amino acid sequence from the Fc region of the parentantibody by from 1 amino acid to 5 amino acids, or from 5 amino acids to10 amino acids. Changes in amino acid sequence are accomplished by oneor more of amino acid substitution, deletion, insertion, etc.

In some embodiments, the parent antibody is a wild-type antibody, e.g.,a naturally-occurring antibody. In other embodiments, the parentantibody is a synthetic antibody, a recombinant antibody, a chimericantibody, etc. In some embodiments, an Fc region modified antibody (“Fcvariant antibody”) is a monoclonal antibody (e.g., an “Fc variant MAb”).

In some embodiments, a subject method of making a set of relatedantibodies comprises one or more of:

a) substituting at least one amino acid residue in the Fc region of aparent antibody, generating an Fc variant antibody, such that the Fcvariant antibody exhibits enhanced in vitro ADCC function to at leastone Fcγ receptor encoded by an Fcγ receptor gene of a first genotype,compared to the FcγR in vitro ADCC function of the parent antibody, togenerate a first Fc variant antibody;

b) substituting at least one amino acid residue in the Fc region of aparent antibody, generating an Fc variant antibody, such that the Fcvariant antibody exhibits enhanced in vitro ADCC function to at leastone Fcγ receptor encoded by an Fcγ receptor gene of a second genotype,compared to the FcγR in vitro ADCC function of the parent antibody, togenerate a second Fc variant antibody;

c) substituting more than one amino acid residue in the Fc region of aparent antibody, generating an Fc variant antibody, such that the Fcvariant antibody exhibits enhanced in vitro ADCC function to both Fcγreceptors encoded by the Fcγ receptor genes, compared to the FcγR invitro ADCC function of the parent antibody;

d) combining the amino acid substitutions of the first and second Fcvariant antibodies to generate a third Fc variant antibody such that thethird Fc variant antibody exhibit enhanced in vitro ADCC function toboth Fcγ receptors encoded by the Fcγ receptor genes, compared to theFcγR in vitro ADCC function of the parent antibody; and

e) generating patient Group-specific Fc variant antibodies such that thedegree of treatment response to antibody therapy in the patient Groupsis increased.

In certain embodiments, first and second Fc variant antibodies have thesame antigen specificity, and have the same antigen specificity as theparent antibody. Antibodies that are members of a set of relatedantibodies include monoclonal antibodies, chimeric antibodies,bi-specific antibodies, recombinant antibodies, synthetic antibodies,semi-synthetic antibodies, tetravalent (multivalent) antibodies,Fc:fusion proteins, and chemically modified intact antibodies.

The parent antibody, the first Fc variant antibody, and the second Fcvariant antibody constitute a set of related antibodies. In certainembodiments, the set of related antibodies are therapeutic antibodiessuitable for treating an ADCC-treatable disease, condition, or disorder.In some embodiments, a set of related therapeutic antibodies areantibodies that bind a tumor-associated antigen; such a set of relatedantibodies is useful for treating an individual having a neoplasticdisease. In other embodiments, a set of related therapeutic antibodiesare antibodies that bind an antigen on an autoreactive immune cell; sucha set of related antibodies is useful for treating an individual havingan autoimmune disorder. In other embodiments, a set of relatedtherapeutic antibodies are antibodies that bind an antigen on analloreactive T-cell; such a set of related antibodies is useful fortreating allograft rejection. In other embodiments, a set of relatedtherapeutic antibodies are antibodies that bind a viral antigenexpressed on the surface of a virus-infected cell; such a set of relatedantibodies is useful for treating a virus infection. In otherembodiments, a set of related therapeutic antibodies are antibodies thatbind an antigen expressed on the surface of a parasite; such a set ofrelated antibodies is useful for treating a parasite infection.

Any convenient method of modifying the amino acid sequence of the Fcregion can be used. In some embodiments, residues that are proximal toresidues that are known to bind or contact an Fc receptor are modified.For example, based on the crystal structure of an Fc portion of anantibody, an amino acid residue that, if substituted, enhances bindingto an Fc receptor, is an amino acid residue that is within about 30 Å,within about 15 Å, within about 10 Å, or within about 5 Å, of an aminoacid residue that is known to contact an Fc receptor. In otherembodiments, residues of the Fc region that are proximal to other Fcresidues on a three-dimensional space, if substituted, enhance bindingto an Fc receptor. Such examples include interactions between T²⁵⁶ andK²⁹⁰, and K³³⁸ and E⁴³° (e.g., Shields et al., supra). These residuesmay be involved in van der Waals contacts, hydrogen bonding, and saltbridge formation.

The Fc fragment including the hinge can be successfully produced as adimer in E. coli (Kim et al., 1994, Eur. J. Immunol. 24:542). SolubleFcγRIII and FcγRII can also be expressed in E. coli (Sondermann andJacob, 1999, Biol. Chem. 380:717-721; Sondermann et al., 2000, Nature406:267-273), and through binding and crystallographic studies it isshown that the Fc fragment binds to FcR domain. Thus, in one embodiment,a) in vitro ADCC is a function of Fc-FcR binding, and b) ADCC-dependenttherapeutic response is a function of in vitro ADCC. In anotherembodiment, in vitro Fc-FcR binding studies and in vitro ADCC assays canbe employed to develop Fc variant MAbs specific for patient Groups I-IXas exemplified in this disclosure based on the differences in FcγRpolymorphisms.

Fc variant antibodies of interest include antibodies that have at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 100%, or 2-fold,increased binding affinity to an Fc receptor and/or enhanced in vitroADCC activity, compared to the binding affinity of the parent antibodyto the Fc receptor and/or in vitro ADCC activity. In some embodiments,Fc variant Abs of interest include antibodies that have at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or at least about 100%, or 2-fold, increasedbinding affinity and/or enhanced in vitro ADCC activity to an FcγRIIAcomprising Arg¹³¹, compared to the binding affinity or ADCC activity ofthe parent antibody to the FcγRIIA comprising Arg¹³¹. In someembodiments, Fc variant Abs of interest include antibodies that have atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 100%, or 2-fold,increased binding affinity and/or enhanced in vitro ADCC activity to anFcγRIIIA comprising Phe¹⁵⁸, compared to the binding affinity or ADCCactivity of the parent antibody to an FcγRIIIA comprising Phe¹⁵⁸.

Fc variant antibodies of interest will comprise one or more amino acidsequence modifications (substitutions, insertions, deletions, etc.) thatprovide for enhanced binding affinity and/or enhanced in vitro ADCCactivity to an FcγRIIA and/or an FcγRIIIA; and will in some embodimentscomprise one or more amino acid substitutions in a hinge region, and/ora CH2 region, and/or a CH3 region. See, e.g., Wines et al., 2000, J.Immunol. 164:5313; Lazar et al., 2006, Proc. Natl. Acad. Sci. USA103:4005-4010. In some embodiments, Fc variant antibodies of interestwill comprise one or more amino acid substitutions in the lower hingeregion, e.g., amino acids L²³⁴-S²³⁹ (the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). The “EUindex as in Kabat” refers to the residue numbering of the human IgG1 EUantibody. In some embodiments, Fc variant antibodies of interest willcomprise one or more amino acid substitutions in one or more of thefollowing regions: R²⁵⁵-T²⁶⁰; D²⁶⁵-E²⁶⁹; N²⁹⁷-T²⁹⁹; A³²⁷-I³³² (See,e.g., FIG. 12-C). In some embodiments, Fc variant antibodies of interestwill comprise one or more amino acid substitutions in the CH3 domain,which when substituted, make considerable improvements in FcR binding(e.g., K³³⁸ and E⁴³⁰).

Fc variant antibodies of interest will comprise one or more amino acidsubstitutions that provide for enhanced binding affinity and/or enhancedin vitro ADCC activity to an FcγRIIA and/or an FcγRIIIA For example, ahuman IgG1 constant region comprising a T256A or a K290A substitutionhas enhanced binding affinity to FcγRIIA and FcγRIIIA, compared towild-type human IgG1. A human IgG1 constant region comprising a E333A,K334A, or A339T substitution has enhanced binding affinity to FcγRIIIA,compared to wild-type human IgG1. Similarly, a human IgG1 constantregion comprising the following triple mutations has enhanced bindingaffinity to FcγRIIIA: S298A, E333A; K334A. See, e.g., Shields et al.,2001, J. Biol. Chem. 276:6591-6604; and Presta et al., 2002, Biochem.Soc. Trans. 30:487-490.

In some embodiments, an Fc variant antibody exhibits enhanced bindingaffinity and/or enhanced in vitro ADCC activity between the Fc region ofthe antibody and an Fcγ receptor, compared to the binding affinity andADCC activity between the Fc region of a parent antibody and the Fcγreceptor. In some embodiments, an Fc variant antibody exhibits an atleast 10%, at least 15%, at least 25%, at least 50%, at least 75%, atleast 100% (or two-fold), at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold, or more, higher affinity and/or enhanced invitro ADCC activity for an Fpγ receptor than the corresponding parentantibody.

In some embodiments, a method of generating an Fc variant antibody thatexhibits enhanced binding affinity and/or enhanced in vitro ADCCactivity to an Fc receptor involves modifying a codon in a nucleic acidthat comprises a nucleotide sequence that encodes an antibody constantregion (including, e.g., hinge region, CH2, and CH3 domains). In someembodiments, a nucleic acid comprising a nucleotide sequence encoding anFc region is amplified by PCR, using a set of primers that encode allnineteen naturally-occurring amino acid variants at a single residue ofthe constant region, to generate a set of variant nucleic acids encodingnineteen amino acid substitution variants at the single residue of theconstant region. Each of the variants is expressed in an in vitrotranscription/translation system, to generate a set of Fc variantantibodies. Each of the Fc variant antibodies is then tested for bindingaffinity to an Fc receptor. The variants are generated using any knownmethod, including, e.g., the method described in U.S. Pat. No.6,180,341. Assessing binding to Fc receptors is carried out using anyknown method, including a method as described in Shields et al., 2001,J. Biol. Chem. 276:6591-6604.

In other embodiments, a method of generating an Fc variant antibody thatexhibits enhanced binding affinity to an Fc receptor involves generatinga set of variant nucleic acids encoding nineteen amino acid substitutionvariants at the single residue of the constant region, as describedabove; expressing each of the encoded Fc variant antibodies on thesurface of a host cell; and selecting a host cell that expresses an Fcvariant antibody that exhibits enhanced binding and/or enhanced in vitroADCC activity to an Fc receptor. See, e.g., U.S. Patent Publication No.2003/0036092.

The present disclosure further provides sets of related antibodies, asdescribed above. In many embodiments, the antibodies are isolated.

Antigen-Binding Specificity

A set of related antibodies includes member antibodies that shareantigen-binding specificity; and that differ in binding affinity to anFcγR. An antibody that is a member of a set of related antibodies willhave an antigen-binding domain; and an Fc domain. Antigen-bindingdomains (ABD) include ABD specific for an epitope on the surface of atumor cell; ABD specific for a virally-encoded epitope on the surface ofa virus-infected cell; ABD specific for an epitope on the surface of anautoreactive cell; ABD specific for an epitope on the surface of analloreactive cell; and ABD specific for an epitope expressed on thesurface of a eukaryotic parasite, e.g., a helminth. An ABD from anyknown antibody can be linked to an Fc fragment, e.g., a native Fcfragment, or a variant Fc fragment that exhibits enhanced binding to oneor more FcγRs, in generating a set of related antibodies.

Sequences of exemplary Fc fragments (e.g., RITUXAN®, REMICADE®, ERBITUX®CAMPATH®, and HERCEPTIN®) are shown in FIG. 15.

In some embodiments, antigen binding domains may be obtained from anyone of the following antibodies: RITUXAN®, CAMPATH®, ZENAPAX®,HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®, REMICADE®, HUMIRA®, ERBITUX®,SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®, MYLOTARG®, REOPRO®, OKT3®,BEXXAR® or ZEVALIN®, ENBREL® or AMEVIVE®

Antigen-binding domains will in some embodiments comprise a V_(H)-C_(H1)region and a V_(L)-C_(L) region. Antigen-binding domains will in someembodiments be a V_(H) or V_(L) domains (domain antibodies), Fv, Fab,scFv, F(ab)₂ fragments, tetravalent (multivalent) antibodies, Fc:fusionproteins, or engineered Fc:fusion constructs (e.g., SMIPs; TrubionPharmaceuticals). The antigen-binding domain will in some embodimentshave the amino acid sequence of an antigen-binding domain of anaturally-occurring antibody. The antigen-binding domain will in someembodiments be recombinant, synthetic, or semi-synthetic. “Humanized”ABD are also suitable for use; see, e.g., U.S. Pat. No. 6,180,370.

Enhanced Antigen Affinity Improves ADCC

The impact of antigen density and antibody affinity on the efficacy oftumor cell elimination via has been studied extensively. Thelow-affinity Ep-CAM MAb 17-1A (K_(a)=5×10⁷ M⁻¹) and the high affinityMAb 323/A3 (K_(a)=2×10⁹ M⁻¹) were compared in ADCC experiments againstmurine and human tumor target cells (Velders et al. supra). Thehigh-affinity MAb mediated ADCC killing of tumor cells with low antigenexpression levels. Even at comparable MAb-binding levels, ADCC mediatedby the high-affinity MAb was more effective. The kinetics of ADCC wasalso found to be determined by the level of antigen expression, and bythe affinity and concentration of the MAb used (Velders et al. supra).

Additionally, the heterogeneity of a target antigen expression is acommon feature of all tumors, and some tumor cells express very lowlevels of antigen. For example, the CD20 antigen density varies invarious NHL types (antigens per cell): normal B-cells, 94,000; mantlecell leukemia, 123,000; pro-lymphocytic leukemia, 129,000; spleniclymphoma, 167,000; hairy cell lymphoma, 312,000; chronic lymphocyticleukemia, 65,000; B-cell (peripheral blood) CLL patients, 9,000; bonemarrow and lymph nodes, 4,000 (Manshouri et al., 2003, Blood 101:2507).

Affinity improvements in antibodies are primarily accomplished throughCDR engineering, and it is a function of association and dissociationrate constants, k_(on), and k_(off) (Schier et al., 1996, J. Mol. Biol.263:551; Wu et al., 1998, Proc. Natl. Acad. Sci. USA 95:6037). Thus,improved ADCC in high-affinity antibodies can be due to a) retention ofMAbs bound to the antigens for such long periods in a conformationallylocked position that facilitates Fc binding to FcγRs, and b) reducedinternalization of antibodies as has been shown for anti-Her2/neuantibody variants (for example, McCall et al., 2001, J Immunol.166:6112; Tang et al., “High affinity promotes more effective ADCC byanti-HER2/neu monocloncal antibodies,” Abstract No: 2538, AmericanSociety of Clinical Oncology, 2006 ASCO Annual Meeting).

In one embodiment, affinity improvement through CDR engineering improvesthe affinity of the IgG Fc region to FcγRIIIA and FcγRIIA. In anotherembodiment, affinity improvement through CDR engineering improves thebinding of the IgG Fc region to FcγRIIIA and FcγRIIA. In yet anotherembodiment, affinity improvement through CDR engineering improves the invitro ADCC mediated by NK cells and/or macrophages. In anotherembodiment, affinity improvement through CDR engineering improves theclinical efficacy of an ADCC-mediated antibody therapy.

Cancer Cell Antigens

Examples of antigens that are expressed on the surface of cancer cellsand to which an ABD binds include, but are not limited to,p185.sup.HER2; CD20; CD19; CD21; CD22; CD23; epidermal growth factor(EGF) receptor; vascular endothelial growth factor (VEGF) receptor;CD33; CD52; epithelial cell adhesion molecule (Ep-CAM); carcinoembryonicantigen (CEA); B-cell idiotypes; CD40; CD80; MHC Class-II; CTLA-4; G250antigen; GD2; and the like. Such antigens are suitable targets for anADCC-based antibody therapy.

The p185.sup.HER2 antigen has been described. See, e.g., Coussens, L. etal., 1985, Science 230:1132-1139; Yamamoto, T. et al., 1986, Nature319:230-234; King, C. R. et al., 1985, Science 229:974-976). HER2 isalso known in the field as c-erbB-2, and sometimes by the name of therat homolog, neu. Amplification and/or overexpression of HER2 isassociated with multiple human malignancies and appears to be integrallyinvolved in progression of 25-30% of human breast and ovarian cancers(Slamon et al., 1987, Science 235:177-182; Slamon et al., 1989, Science244:707-712). Furthermore, the extent of amplification is inverselycorrelated with the observed median patient survival time (Slamon,supra). Antibodies that bind HER2 are known in the art, and an ABD ofany known anti-HER2 antibody can be used. The murine monoclonal antibodyknown as muMAb4D5 (Fendly et al., 1990, Cancer Res. 50:1550-1558),directed against the extracellular domain (ECD) of p185.sup.HER2,specifically inhibits the growth of tumor cell lines overexpressingp185.sup.HER2 in monolayer culture or in soft agar (Hudziak et al.,1989, Mol. Cell. Biol. 9:1165-1172; Lupu et al., 1990, Science249:1552-1555). MuMAb4D5 also has the potential of enhancing tumor cellsensitivity to tumor necrosis factor, an important effector molecule inmacrophage-mediated tumor cell cytotoxicity (Hudziak, supra, 1989;Shepard, H. M. and Lewis, G. D., 1988, J. Clinical Immunology8:333-395). The ABD of muMAb4D5 is useful in the treatment of cancercells in which p185.sup.HER2 is overexpressed. The muMAb4D5 antibody isdescribed in PCT application WO 89/06692 published 27 Jul. 1989. Thismurine antibody was deposited with the ATCC and designated ATCC CRL10463. A “humanized” version of MAb4D5 is discussed in U.S. Pat. No.6,800,738.

Antibodies to CD20 are known in the art and include Rituximab (asdiscussed above). Antibodies to CD52 include, e.g., Alemtuzumab(Campath-1H) (see, e.g., U.S. Pat. No. 5,545,403). Antibodies to CD33include, e.g., gemtuzumab (Myelotarg); humanized M195 (Caron et al.,1994, Blood 83:1760-1768; and Co et al., 1992, J. Immunol. 148:1149).Antibodies to EGF receptor include, e.g., cetuximab (Erbitux); andpanitumumab (Abgenix/Immunex). Antibodies to GD2 (expressed onneuroblastoma cells) include, e.g., Ch14.18 (NCI). Antibodies to G250antigen (expressed on renal cancer cells) include, e.g., Rencarex(WX-9250; Wilex). Antibodies to MHC class-II (expressed on non-Hodgkinslymphoma cells) include, e.g., Remitogen (Hu1D10; Protein Design Labs).

Autoreactive Immune Cell Antigens

Antigens expressed on the surface of autoreactive immune cells that aresuitable as targets of an ADCC-based antibody therapy include, but arenot limited to, CD3.

Antigens Expressed on Alloreactive T-Cells

Antigens on alloreactive T-cells that are suitable as targets of anADCC-based antibody therapy include, but are not limited to, CD3, CD2,CD4, CD5, CD6, CD8, CD28, and CD44.

Microbial Antigens

Viral antigens that are suitable as targets of an ADCC-based antibodytherapy include virus-encoded antigens expressed on the surface of avirus-infected cell. Viral antigens are from any of a variety of DNA andRNA viruses. Although HCV is primarily a hepatotropic virus, HCVinfection is frequently associated with mixed cryoglobulinemia,non-Hodgkin's B-cell lymphoma, and Sjogren's syndrome, all of whichinvolve B-cell proliferation (Selva-O'Callaghan et. al., 1999, ArthritisRheum. 42:2489), suggesting that HCV may infect B-cells or affect B-cellfunctions in natural infection. Minus-strand HCV RNA has been detectedby reverse transcriptase PCR in peripheral lymphocytes, bone marrow,lymph nodes, and central nervous system of some HCV patients (Radkowskiet. al., 2002, J. Virol. 76:600).

Parasitic antigens that are suitable targets for ADCC-based antibodytherapy include antigens expressed on the surface of a parasite that isthe etiologic agent of a parasitic disorder. Bacterial antigens that aresuitable targets for ADCC-based antibody therapy include antigensexpressed on the surface of a bacterium that is the etiologic agent of abacterial infection.

Preparation of Antibodies

Variant antibodies are produced by modifying the nucleic acid of aparent antibody, inserting the modified nucleic acid into an appropriatecloning vector and expressing the modified nucleic acid to producevariant antibodies. Representative protocols are described below:

1. Making Variant Antibody Nucleic Acid

Variant antibodies will comprise one or more amino acid sequencemodifications (substitutions, insertions, deletions, etc.) relative to aparent antibody sequence that provide for enhanced binding affinityand/or enhanced in vitro ADCC activity to an FcγRIIA and/or an FcγRIIIA.

In some embodiments, an Fc variant antibodies will comprise one or moreamino acid substitutions in a hinge region, and/or a CH2 region, and/ora CH3 region (See, e.g., Wines et al., 2000, J. Immunol. 164:5313; Lazaret al., 2006, Proc. Natl. Acad. Sci. USA 103:4005-4010). In otherembodiments, Fc variant antibodies of interest will comprise one or moreamino acid substitutions in the lower hinge region, e.g., amino acidsL²³⁴-S²³⁹ (the numbering of the residues in an immunoglobulin heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody. In yetfurther embodiments, Fc variant antibodies of interest will comprise oneor more amino acid substitutions in one or more of the followingregions: R²⁵⁵-T²⁶⁰; D²⁶⁵-E²⁶⁹; N²⁹⁷-T²⁹⁹; A³²⁷-I³³² (See, e.g., FIG.12-C). In some embodiments, Fc variant antibodies of interest willcomprise one or more amino acid substitutions in the CH3 domain, whichwhen substituted, make considerable improvements in FcR binding (e.g.,K³³⁸ and E⁴³⁰).

Fc variant antibodies of interest will comprise one or more amino acidsubstitutions that provide for enhanced binding affinity and/or enhancedin vitro ADCC activity to an FcγRIIA and/or an FcγRIIIA. For example, ahuman IgG1 constant region comprising a T256A or a K290A substitutionhas enhanced binding affinity to FcγRIIA and FcγRIIIA, compared towild-type human IgG1. A human IgG1 constant region comprising a E333A,K334A, or A339T substitution has enhanced binding affinity to FcγRIIIA,compared to wild-type human IgG1. Similarly, a human IgG1 constantregion comprising the following triple mutations has enhanced bindingaffinity to FcγRIIIA: S298A, E333A; K334A (See, e.g., Shields et al.,2001, J. Biol. Chem. 276:6591-6604; and Presta et al., 2002, Biochem.Soc. Trans. 30:487-490).

In some embodiments, an Fc variant antibody exhibits enhanced bindingaffinity and/or enhanced in vitro ADCC activity between the Fc region ofthe antibody and an Fcγ receptor, compared to the binding affinity andADCC activity between the Fc region of a parent antibody and the Fcγreceptor. In some embodiments, an Fc variant antibody exhibits an atleast 10%, at least 15%, at least 25%, at least 50%, at least 75%, atleast 100% (or two-fold), at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold, or more, higher affinity and/or enhanced invitro ADCC activity for an Fγ receptor than the corresponding parentantibody.

In some embodiments, a method of generating an Fc variant antibody thatexhibits enhanced binding affinity and/or enhanced in vitro ADCCactivity to an Fc receptor involves modifying a codon in a nucleic acidthat comprises a nucleotide sequence that encodes an antibody constantregion (including, e.g., hinge region, CH2, and CH3 domains). In someembodiments, a nucleic acid comprising a nucleotide sequence encoding anFc region is amplified by PCR, using a set of primers that encode allnineteen naturally-occurring amino acid variants at a single residue ofthe constant region, to generate a set of variant nucleic acids encodingnineteen amino acid substitution variants at the single residue of theconstant region.

The antibody variants are generated using any known method, including,e.g., the method described in U.S. Pat. No. 6,180,341.

In other embodiments, a method of generating an Fc variant antibody thatexhibits enhanced binding affinity to an Fc receptor involves generatinga set of variant nucleic acids encoding nineteen amino acid substitutionvariants at the single residue of the constant region, as describedabove; expressing each of the encoded Fc variant antibodies on thesurface of a host cell; and selecting a host cell that expresses an Fcvariant antibody that exhibits enhanced binding and/or enhanced in vitroADCC activity to an Fc receptor (See, e.g., U.S. Patent Publication No.2003/0036092).

In other embodiments, antibody variants may comprise one or more aminoacid substitutions in a complementarity determining region (CDR). Suchmodifications may be generated by any of the methods describe aboveconcerning Fc variants.

In designing amino acid sequence antibody variants, the location of themutation site and the nature of the mutation will depend on the targetantibody characteristic(s) to be modified. The sites for mutation can bemodified individually or in series, e.g., by (1) substituting first withconservative amino acid choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue, or(3) inserting residues of the same or a different class adjacent to thelocated site, or combinations of options 1-3.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells, 1989,Science, 244: 1081-1085. Here, a residue or Group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with the surrounding aqueous environment in or outside thecell. Those domains demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis may be conducted at the target codon or region andthe expressed antibody variants are screened for the optimal combinationof desired activity.

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. In general, the location and nature of the mutation chosenwill depend upon the antibody characteristic to be modified.

Amino acid sequence deletions of antibodies are generally not preferred,as maintaining the generally configuration of an antibody is believed tobe necessary for its activity. Any deletions will be selected so as topreserve the structure of the antibody.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the antibody sequence) may range generally from about1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.Examples of terminal insertions include the antibody with an N-terminalmethionyl residue, an artifact of the direct expression of targetpolypeptide in bacterial recombinant cell culture, and fusion of aheterologous N-terminal signal sequence to the N-terminus of the targetpolypeptide molecule to facilitate the secretion of the mature targetpolypeptide from recombinant host cells. Such signal sequences generallywill be obtained from, and thus homologous to, the intended host cellspecies. Suitable sequences include STII or Ipp for E. coli, alphafactor for yeast, and viral signals such as herpes gD for mammaliancells.

Another group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of the antibody, and sites where theamino acids found in the antibody from various species are substantiallydifferent in terms of side-chain bulk, charge, and/or hydrophobicity.Other sites for substitution are described infra, considering the effectof the substitution of the antigen binding, affinity and othercharacteristics of a particular target antibody.

Other sites of interest are those in which particular residues of theantibody obtained from various species are identical. These positionsmay be important for the biological activity of the antibody. Thesesites, especially those falling within a sequence of at least threeother identically conserved sites, are substituted in a relativelyconservative manner. If such substitutions result in a change inbiological activity, then other changes are introduced and the productsscreened until the desired effect is obtained.

Substantial modifications in function or immunological identity of theantibody are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into Groups based on commonside chain properties:

-   -   hydrophobic: norleucine, met, ala, val, leu, ile;    -   neutral hydrophilic: cys, ser, thr;    -   acidic: asp, glu;    -   basic: asn, gin, his, lys, arg;    -   residues that influence chain orientation: gly, pro; and    -   aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues may be introducedinto regions of the antibody that are homologous with other antibodiesof the same class or subclass, or, more preferably, into thenon-homologous regions of the molecule.

Any cysteine residues not involved in maintaining the properconformation of the antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking.

DNA encoding amino acid sequence variants of the antibody are preparedby a variety of methods known in the art. These methods include, but arenot limited to, isolation from a natural source (in the case ofnaturally occurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the target polypeptide. A particularlypreferred method of gene conversion mutagenesis is described below inExample 1. These techniques may utilized antibody nucleic acid (DNA orRNA), or nucleic acid complementary to the antibody nucleic acid.Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of antibody DNA. Thistechnique is well known in the art as described by Adelman et al., 1983,DNA, 2: 183. Briefly, the antibody DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of the targetpolypeptide. After hybridization, a DNA polymerase is used to synthesizean entire second complementary strand of the template that will thusincorporate the oligonucleotide primer, and will code for the selectedalteration in the target polypeptide DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., 1978,Proc. Natl. Acad. Sci. USA, 75: 5765.

Single-stranded DNA template may also be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the antibody, and the other strand (the original template)encodes the native, unaltered sequence of the antibody. Thisheteroduplex molecule is then transformed into a suitable host cell,usually a prokaryote such as E. coli JM 101. After the cells are grown,they are plated onto agarose plates and screened using theoligonucleotide primer radiolabeled with 32-phosphate to identify thebacterial colonies that contain the mutated DNA. The mutated region isthen removed and placed in an appropriate vector for protein production,generally an expression vector of the type typically employed fortransformation of an appropriate host. The method described immediatelyabove may be modified such that a homoduplex molecule is created whereinboth strands of the plasmid contain the mutation(s). The modificationsare as follows: The single-stranded oligonucleotide is annealed to thesingle-stranded template as described above. A mixture of threedeoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine(dGTP), and deoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex.

Upon addition of DNA polymerase to this mixture, a strand of DNAidentical to the template except for the mutated bases is generated. Inaddition, this new strand of DNA will contain dCTP-(aS) instead of dCTP,which serves to protect it from restriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding antibody variants with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant antibody. The first round is as described forthe single antibody mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid antibodyvariants. While the following discussion refers to DNA, it is understoodthat the technique also finds application with RNA. The PCR techniquegenerally refers to the following procedure (See, e.g., Erlich, supra,the chapter by R. Higuchi, p. 61-70). When small amounts of template DNAare used as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer is located within 200 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide tri-phosphates andis included in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayed with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/4 purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 min.at 55° C., then 30 sec. at 72° C., then 19 cycles of the following: 30sec. at 94° C., 30 sec. at 55° C., and 30 sec. at 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50:vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., 1985, Gene, 34:315. Thestarting material is the plasmid (or other vector) comprising theantibody DNA to be mutated. The codon(s) in the antibody DNA to bemutated are identified. There must be a unique restriction endonucleasesite on each side of the identified mutation site(s). If no suchrestriction sites exist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the target polypeptide DNA. After therestriction sites have been introduced into the plasmid, the plasmid iscut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated antibody DNA sequence.

2. Insertion of DNA into a Cloning Vehicle

The cDNA or genomic DNA encoding the antibody is inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. Many vectors are available, and selection of the appropriatevector will depend on 1) whether it is to be used for DNA amplificationor for DNA expression, 2) the size of the DNA to be inserted into thevector, and 3) the host cell to be transformed with the vector. Eachvector contains various components depending on its function(amplification of DNA or expression of DNA) and the host cell for whichit is compatible. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(a) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of antibody DNA that is inserted into the vector.

The therapeutic antibody may be expressed not only directly, but also asa fusion with a heterologous polypeptide, preferably a signal sequenceor other polypeptide having a specific cleavage site at the N-terminusof the mature protein or polypeptide. In general, the signal sequencemay be a component of the vector, or it may be a part of the antibodyDNA that is inserted into the vector. Included within the scope of thisdisclosure are antibody with any native signal sequence deleted andreplaced with a heterologous signal sequence. The heterologous signalsequence selected should be one that is recognized and processed (i.e.cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process the native antibody signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.For yeast secretion the native target polypeptide signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

(b) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is, transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus as a host, for example, by includingin the vector a DNA sequence that is complementary to a sequence foundin Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theantibody DNA. However, the recovery of genomic DNA encoding the antibodyis more complex than that of an exogenously replicated vector becauserestriction enzyme digestion is required to excise the antibody DNA.

(c) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., 1982, J. Molec. Appl. Genet.1:327), mycophenolic acid (Mulligan et al., 1980, Science, 209:1422) orhygromycin (Sugden et al., 1985, Mol. Cell. Biol., 5:410-413). The threeexamples given above employ bacterial genes under eukaryotic control toconvey resistance to the appropriate drug G418 or neomycin (geneticin),xgpt (mycophenolic acid), or hygromycin, respectively. Another exampleof suitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take up the antibody nucleicacid, such as dihydrofolate reductase (DHFR) or thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesantibody. Amplification is the process by which genes in greater demandfor the production of a protein critical for growth are reiterated intandem within the chromosomes of successive generations of recombinantcells. Increased quantities of the antibody are synthesized from theamplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, 1980, Proc. Natl. Acad.Sci. USA, 77:4216. The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding theantibody.

This amplification technique can be used with any otherwise suitablehost, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060). Alternatively, host cells(particularly wild-type hosts that contain endogenous DHFR) transformedor co-transformed with DNA sequences encoding the antibody, wild-typeDHFR protein, and another selectable marker such as aminoglycoside 3′phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418 (See,e.g., U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature, 282: 39;Kingsman et al., 1979, Gene, 7:141; or Tschemper et al., 1980, Gene,10:157). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, 1977, Genetics, 85:12). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(d) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of a particularnucleic acid sequence, such as that encoding the antibody, to which theyare operably linked. Such promoters typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from DNA under their controlin response to some change in culture conditions, e.g., the presence orabsence of a nutrient or a change in temperature. At this time a largenumber of promoters recognized by a variety of potential host cells arewell known. These promoters are operably linked to DNA encoding thetarget polypeptide by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native antibody promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the antibody DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of expressed antibody as compared to the native antibodypromoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., 1978, Nature,275: 615; and Goeddel et al., 1979, Nature, 281: 544), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, 1980, NucleicAcids Res., 8: 4057 and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., 1983, Proc. Natl. Acad. Sci. USA, 80:21-25).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the target polypeptide(Siebenlist et al., 1980, Cell, 20:269) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also generally will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the target polypeptide.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980, J. Biol.Chem. 255:2073) or other glycolytic enzymes (Hess et al, 1968, J. Adv.Enzyme Reg. 7:149; and Holland, 1978, Biochemistry 17:4900), suchasenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, met allothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

Antibody transcription from vectors in mammalian host cells iscontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with theantibody sequence, provided such promoters are compatible with the hostcell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., 1978, Nature, 273:113; Mulligan and Berg,1980, Science, 209: 1422-1427; Pavlakis et al., 1981, Proc. Natl. Acad.Sci. USA, 78: 7398-7402. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., 1982, Gene, 18: 355-360. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See, e.g., Gray et al.,1982, Nature, 295: 503-508 on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., 1982, Nature, 297: 598-601 on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus, Canaani and Berg,1982, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 on expression of thehuman interferon-131 gene in cultured mouse and rabbit cells, and Gormanet al., 1982, Proc. Natl. Acad. Sci. USA, 79: 6777-6781 on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

(e) Enhancer Element Component

Transcription of DNA encoding the antibody of this disclosure by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from10-300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5′ (Laimins et al., 1981, Proc. Natl. Acad. Sci. USA 78:993)and 3′ (Lusky et al., 1983, Mol. Cell. Bio. 3:1108) to the transcriptionunit, within an intron (Banerji et al., 1983, Cell 33:729) as well aswithin the coding sequence itself (Osborne et al., 1984, Mol. Cell. Bio.4:1293). Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, -fetoprotein and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See, e.g., Yaniv, 1982, Nature 297:17-18 on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibody DNA, butis preferably located at a site 5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNA or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the target polypeptide. The 3′ untranslatedregions also include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components the desired coding and control sequences employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., 1981,Nucleic Acids Res. 9:309 or by the method of Maxam et al., 1980, Methodsin Enzymology. 65:499.

Particularly useful in the practice of this disclosure are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the antibody. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredantibody encoded by the expression vector. Transient expression systems,comprising a suitable expression vector and a host cell, allow for theconvenient positive identification of polypeptides encoded by clonedDNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the disclosure forpurposes of identifying analogs and variants of the antibody that haveantibody-like activity.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the target polypeptide in recombinant vertebrate cellculture are described in Gething et al., 1981, Nature 293:620-625;Mantei et al., 1979, Nature 281:40-46; Levinson et al.; EP 117,060; andEP 117,058. A particularly useful plasmid for mammalian cell cultureexpression of the antibody is pRK5 (EP pub. no. 307,247) or pSVI6B.Selection and

3. Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescens. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli.chi.1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.

These examples are illustrative rather than limiting. Preferably thehost cell should secrete minimal amounts of proteolytic enzymes.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for target polypeptide-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe (Beach and Nurse, 1981,Nature 290:140; EP 139,383 published May 2, 1985, Kluyveromyces hosts(U.S. Pat. No. 4,943,529) such as, e.g., K. lactis (Louvencourt et al.,1983, J. Bacteriol., 737), K. fragilis, K. bulgaricus, K.thermotolerans, and K. marxianus, yarrowia (EP 402,226), Pichia pastoris(EP 183,070; Sreekrishna et al., 1988, J. Basic Microbiol., 28:265-278), Candida, Trichoderma reesei (EP 244,234), Neurospora crassa(Case et al., 1979, Proc. Natl. Acad. Sci. USA, 76: 5259-5263), andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published Jan. 10, 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., 1983, Biochem. Biophys. Res. Commun.112:284-289); Tilburn et al., 1983, Gene 26:205-221); Yelton et al.,1984, Proc. Natl. Acad. Sci. USA 81:1470-1474) and A. niger (Kelly andHynes, 1985, EMBO J. 4:475-479).

Suitable host cells for the expression of glycosylated targetpolypeptide are derived from multicellular organisms. Such host cellsare capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori host cells have been identified. See, e.g., Luckow et al., 1988,Bio/Technology 6:47-55; Miller et al., in Genetic Engineering, Setlow,J. K. et al., eds., Vol. 8, Plenum Publishing, pp. 277-279 (1986); andMaeda et al., 1985, Nature 315:592-594). A variety of such viral strainsare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present disclosure, particularlyfor transfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can beutilized as hosts. Typically, plant cells are transfected by incubationwith certain strains of the bacterium Agrobacterium tumefaciens, whichhas been previously manipulated to contain the antibody DNA. Duringincubation of the plant cell culture with A. tumefaciens, the DNAencoding antibody is transferred to the plant cell host such that it istransfected, and will, under appropriate conditions, express theantibody DNA. In addition, regulatory and signal sequences compatiblewith plant cells are available, such as the nopaline synthase promoterand polyadenylation signal sequences. See, Depicker et al., 1982, J.Mol. Appl. Gen. 1:561. In addition, DNA segments isolated from theupstream region of the T-DNA 780 gene are capable of activating orincreasing transcription levels of plant-expressible genes inrecombinant DNA-containing plant tissue. See, e.g., EP 321,196 publishedJun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years (Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., 1977, J. Gen Virol. 36:59); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA77:4216); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad.Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma cell line(Hep G2). Preferred host cells are human embryonic kidney 293 andChinese hamster ovary cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this disclosure andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., 1983, Gene 23:315 and WO 89/05859 publishedJun. 29, 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in sections 16.30-16.37 ofSambrook et al., supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., 1977, J.Bact. 130:946 and Hsiao et al., 1979, Proc. Natl. Acad. Sci. USA76:3829. However, other methods for introducing DNA into cells such asby nuclear injection, electroporation, or protoplast fusion may also beused. Culturing the Host Cells

Prokaryotic cells used to produce the target polypeptide of thisdisclosure are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the target polypeptide of thisdisclosure may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma)are suitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, 1979, Meth. Enz. 58:44; Barnes and Sato,1980, Anal. Biochem. 102:255; U.S. Pat. No. 4,767,704; U.S. Pat. No.4,657,866; U.S. Pat. No. 4,927,762; or U.S. Pat. No. 4,560,655; WO90/03430; WO 87/00195; U.S. Pat. Re. No. 30,985, may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

It is further envisioned that the antibody of this disclosure may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the antibody currently in use in the field. Forexample, a powerful promoter/enhancer element, a suppressor, or anexogenous transcription modulatory element is inserted in the genome ofthe intended host cell in proximity and orientation sufficient toinfluence the transcription of DNA encoding the desired antibody. Thecontrol element does not encode the antibody of this disclosure, but theDNA is present in the host cell genome. One next screens for cellsmaking the antibody of this disclosure, or increased or decreased levelsof expression, as desired.

Enhancement of Antibody Effector Functions

Methods are provided for using a subject's FcγRIIA and/or FcγRIIIAgenotype to select a specific Fc nucleotide sequence associated withoptimal effector function for an antibody therapy.

It is contemplated that the Fc nucleotide sequence from an antibody,including, for example, a variant antibody with one or more optimaleffector functions for a particular FcγRIIA and FcγRIIIA genotype can beused to optimize one or more effector functions of other antibodies usedto treat the same or other subjects with the FcγRIIA and FcγRIIIAgenotype.

In a preferred embodiment, the effector function is ADCC. In otherembodiments, the effector function is selected from the Group consistingof: phagocytosis, opsonization, opsonophagocytosis, Clq binding, andcomplement dependent cell mediated cytotoxicity (CDC).

Generally, methods are provided for enhancing one or more effectorfunctions of an antibody used to treat a subject having anADCC-treatable disease or disorder, comprising: a) genotyping thesubject for an FcγRIIA polymorphism and an FcγRIIIA polymorphism; b)classifying the subject into one of more than three categories of ADCCactivity for the antibody based on their FcγRIIA polymorphism andFcγRIIIA polymorphism; and c) selecting an Fc nucleotide sequence thathas at least one optimized effector function for the FcγRIIApolymorphism and FcγRIIIA polymorphism, wherein at least one effectorfunction of the antibody is enhanced by using the optimized Fcnucleotide sequence.

Fc cassettes are optimized for each of the nine distinct FcγRIIA andFcγRIIIA genotypes, including: V/V¹⁵⁸, H/H¹³¹ (Group-I); V/F¹⁵⁸, H/H¹³¹(Group-II); F/F¹⁵⁸, H/H¹³¹ (Group-III); V/V¹⁵⁸, H/R¹³¹ (Group-IV);V/F¹⁵⁸, H/R¹³¹ (Group-V); F/F¹⁵⁸, H/R¹³¹ (Group-VI); V/V¹⁵⁸, R/R¹³¹(Group-VII); V/F¹⁵⁸, R/R¹³¹ (Group-VIII); and F/F¹⁵⁸, R/R¹³¹ (Group-IX)as previously described in the above methods.

Antibody effector functions, such as ADCC, may be optimized for aparticular FcγRIIA and FcγRIIIA genotype by altering the nucleotidesequence of the Fc portion of the antibody, to an Fc nucleotide sequenceassociated with optimal effector functions for the genotype. Thenucleotide sequence of the Fc region of an antibody is engineered bytechniques commonly known in the art to derive the same nucleotidesequence of Fc that has optimized ADCC activity for a subject with aparticular FcγRIIA and FcγRIIIA genotype.

In other embodiments, the ADCC activity of an antibody used to treat asubject with a particular FcγRIIA and FcγRIIIA genotype can be optimizedby fusing an Fc nucleotide sequence from another antibody molecule whichhas optimized effector functions for the given genotype to the antibody.

Therapeutic antibodies used to treat a particular γRIIA (H/R¹³¹)FcγRIIIA (V/F¹⁵⁸) genotype may be modified to exhibit optimal ADCCactivity. For instance the therapeutic antibody RITUXAN™ has optimalADCC activity for subjects exhibiting a V/V¹⁵⁸, H/H¹³¹ genotype. Hence,the Fc nucleotide sequence of RITUXAN® may be used to optimize ADCCactivity of other antibodies used to treat subjects exhibiting a V/V¹⁵⁸,H/H¹³¹ genotype. For instance, the Fc nucleotide sequence of RITUXAN®may be used to in place of the Fc nucleotide sequence present inZENAPAX® to optimize ADCC activity in subjects exhibiting a V/V¹⁵⁸,H/H¹³¹ genotype.

Examples of other therapeutic antibodies, variable regions of anantibody, or Fc variant antibodies that can be engineered to haveenhanced ADCC activity for a particular FcγRIIA and FcγRIIIA genotype,include but are not limited to: RITUXAN®, CAMPATH®, ZENAPAX®,HERCEPTIN®, XOLAIR®, RAPTIVA®, AVASTIN®, REMICADE®, HUMIRA®, ERBITUX®,SIMULECT®, SYNAGIS®, VECTIBIX®, TYSABRI®, MYLOTARG®, REOPRO®, OKT3®,BEXXAR® or ZEVALIN®.

Optimized fusion antibodies can be produced by standard recombinant DNAtechniques or by protein synthetic techniques, e.g., by use of a peptidesynthesizer. For example, a nucleic acid molecule encoding an optimizedantibody fusion can be synthesized by conventional techniques includingautomated DNA synthesizers.

Alternatively, PCR amplification of gene fragments (i.e. the optimizedFc and antigen binding domain) can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (See, e.g., Current Protocols in MolecularBiology, Ausubel et al., eds., John Wiley & Sons, 1992).

Moreover, a nucleic acid encoding an antigen binding domain can becloned into an expression vector containing an genotype optimized Fcregion such that the antigen binding domain is linked in-frame to theoptimized Fc region.

Methods for fusing or conjugating antigen binding domains to a genotypeoptimized Fc are known in the art (See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181,5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367, 166; EP394,827; International Publication Nos. WO 91/06570, WO 96/04388, WO96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., 1991, Proc.Natl. Acad. Sci. USA 88:10535-10539; Traunecker et al., 1988, Nature331:84-86; Zheng et al., 1.995, J. Immunol. 154:5590-5600; and Vil etal., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341).

The nucleotide sequences encoding a antigen binding domain and an Fcdomain may be obtained from any information available to those of skillin the art (i.e., from Genbank, the literature, or by routine cloning)(See, e.g., Xiong et al., 2001, Science 294(5541):339-45). Thenucleotide sequence coding for an antibody fusion protein can beinserted into an appropriate expression vector, i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence. A variety of host-vector systemsmay be utilized in the present disclosure to express the protein-codingsequence. These include but are not limited to mammalian cell systemsinfected with virus (e.g., vaccinia virus, adenovirus, etc.); insectcell systems infected with virus (e.g., baculovirus); microorganismssuch as yeast containing yeast vectors; or bacteria transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elementsof vectors vary in their strengths and specificities. Depending on thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

Functional Assays for Antibodies

Antibodies, including, for example, variant antibodies may becharacterized in a variety of ways. For example, antibody variants maybe assayed for the ability to specifically bind to a ligand, (e.g.,FcγRIIIA, FcγRIIB, Clq). Such an assay may be performed in solution(e.g., Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991,Nature 354:82-84), on chips (Fodor, 1993, Nature 364:555-556), onbacteria (U.S. Pat. No. 5,223,409), 011 plasmids (Cull et al., 1992,Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith,1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla etal., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J.Mol. Biol. 222:301-310). Molecules that have been identified tospecifically bind to a ligand, (e.g., FcγRIIIA, FcγRIIB, Clq or to anantigen) can then be assayed for their affinity for the ligand.

Antibody variants may be assayed for specific binding to a molecule suchas an antigen (e.g., cancer antigen and cross-reactivity with otherantigens) or a ligand (e.g., FcγR) by any method known in the art,Immunoassays which can be used to analyze specific binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (See, e.g., Ausubel etal., eds, Current Protocols in Molecular Biology, Vol. 1, John Wiley &Sons, Inc., New York (1994).

The binding affinity of the antibody variants to a molecule such as anantigen or a ligand, (e.g., FcγR) and the off-rate of the interactioncan be determined by competitive binding assays. One example of acompetitive binding assay is a radioimmunoassay comprising theincubation of labeled ligand, such as FcγR (e.g., ³H or ¹²⁵1) with amolecule of interest (e.g., antibody variants) in the presence ofincreasing amounts of unlabeled ligand, such as FcγR, and the detectionof the molecule bound to the labeled ligand. The affinity of themolecule of the present disclosure for the ligand and the bindingoff-rates can be determined from the saturation data by scatchardanalysis.

The kinetic parameters of an antibody variant may also be determinedusing any surface plasmon resonance (SPR) based assays known in the art(e.g., BIAcore kinetic analysis). For a review of SPR-based technology,See, e.g., Mullet et al., 2000, Methods 22:77-91; Dong et al., 2002,Review in Mol. Biotech., 82:303-23; Fivash et al., 1998, Current Opinionin Biotechnology 9:97-101; Rich et al., 2000, Current Opinion inBiotechnology 11:54-61.

Additionally, any of the SPR instruments and SPR based methods formeasuring protein-protein interactions described in U.S. Pat. Nos.6,373,577; 6,259,286 5,322,798; 5,341,215; 6,268,125 are contemplated inthe methods of the disclosure.

Fluorescence activated cell sorting (FACS), using any of the techniquesknown to those skilled in the art, can be used for characterizing thebinding of antibody variants to a molecule expressed on the cell surface(e.g., FcγRIIA, FcγRIIIA) Flow sorters are capable of rapidly examininga large number of individual cells that contain library inserts (e.g.,10-100 million cells per hour) (Shapiro et al, Practical Flow cytometry,1995). Flow cytometers for sorting and examining biological cells arewell known in the art. Known flow cytometers are described, for example,in U.S. Pat. Nos. 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796;and 6,211,477. Other known flow cytometers are the FACS Vantage™ systemmanufactured by Becton Dickinson and Company, and the COPAS™ systemmanufactured by Union Biometrica.

The antibody variants can be characterized by their ability to mediateFcγR-mediated effector cell function. Examples of effector cellfunctions that can be assayed include, but are not limited to,antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis,opsonization, opsonophagocytosis, Clq binding, and complement dependentcell mediated cytotoxicity (CDC).

Any cell-based or cell free assay known to those skilled in the art fordetermining effector cell function activity can be used (For effectorcell assays, See, e.g., Perussia et al., 2000, Methods Mol. Biol.121:179-92; Baggiolini et al., 1998, Experientia 44(10): 841-8; Lehmannet al., 2000, J. Immunol, Methods 243(1-2):229-42; Brown E J., 1994,Methods Cell Biol. 45:147-64; Munn et al., 1990, J. Exp. Med.172:231-237, Abdul-Majid et al., 2002, Scand. J. Immunol. 55:70-81; Dinget al., 1998, Immunity 8:403-411).

For example, the antibody variants can be assayed for FcγR-mediated ADCCactivity in effector cells, (e.g., natural killer cells) using any ofthe standard methods known to those skilled in the art (See e.g.,Perussia et al., 2000, Methods Mol. Biol.).

An exemplary assay for determining ADCC activity of the molecules of thedisclosure is based on a ⁵¹Cr release assay comprising of: labelingtarget cells with Na₂CrO₄ (this cell-membrane permeable molecule iscommonly used for labeling since it binds cytoplasmic proteins andalthough spontaneously released from the cells with slow kinetics, it isreleased massively following target cell necrosis); osponizing thetarget cells with the antibody variants of the disclosure; combining theopsonized radiolabeled target cells with effector cells in a microtitreplate at an appropriate ratio of target cells to effector cells;incubating the mixture of cells for 16-18 hours at 37° C.; collectingsupernatants; and analyzing radioactivity. The cytotoxicity of themolecules of the disclosure can then be determined, for example usingthe following formula: % lysis(experimental cpm−target leakcpm)/(detergent lysis cpm−target leak cpm)×100%. Alternatively, %lysis=(ADCC−AICC)/(maximum release-spontaneous release). Specific lysiscan be calculated using the formula: specific lysis % lysis with themolecules of the disclosure−% lysis in the absence of the molecules ofthe disclosure. A graph can be generated by varying either the target:effector cell ratio or antibody concentration.

Methods to characterize the ability of the antibody variants to bind Clqand mediate complement dependent cytotoxicity (CDC) are well known inthe art. For example, to determine Clq binding, a Clq binding ELISA maybe performed. An exemplary assay may comprise the following: assayplates may be coated overnight at 4° C. with polypeptide variant orstarting polypeptide (control) in coating buffer. The plates may then bewashed and blocked. Following washing, an aliquot of human Clq may beadded to each well and incubated for 2 hrs at room temperature.Following a further wash, 100 μL of a sheep anti-complement Clqperoxidase conjugated antibody may be added to each well and incubatedfor 1 hour at room temperature. The plate may again be washed with washbuffer and 100 μl of substrate buffer containing OPD (O-phenylenediaminedihydrochloride (Sigma)) may be added to each well. The oxidationreaction, observed by the appearance of a yellow color, may be allowedto proceed for 30 minutes and stopped by the addition of 100 μl of 4.5NH₂SO₄. The absorbance may then read at (492-405) nm. Specific methodsare also disclosed in the section entitled “Examples,” infra.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, (e.g., as described in Gazzano-Santoro etal., 1996, J. Immunol. Methods 202:163). Briefly, various concentrationsof antibody variant and human complement may be diluted with buffer.Cells which express the antigen to which the Fc variant binds may bediluted to a density of about I &gt; 10 cells/ml. Mixtures of the Fcvariant, diluted human complement and cells expressing the antigen maybe added to a flat bottom tissue culture 96 well plate and allowed toincubate for 2 hrs at 37° C. and 5% CO₂ to facilitate complementmediated cell lysis. 50 μL of alamar blue (Accumed International) maythen be added to each well and incubated overnight at 37° C. Theabsorbance is measured using a 96-well fluorometer with excitation at530 nm and emission at 590 nm. The results may be expressed in relativefluorescence units (RFU). The sample concentrations may be computed froma standard curve and the percent activity, relative to a comparablemolecule.

Complement assays may be preformed with guinea pig, rabbit or humanserum. Complement lysis of target cells maybe detected by monitoring therelease of intracellular enzymes such as lactate dehydrogenase (LDH), asdescribed in Korzeniewski et al., 1983. Immunol. Methods 64(3):313-20;and Decker et at., 1988, Immunol. Methods 115(1):61-9; or the release ofan intracellular label such as europium, chromium 51 or indium 111 inwhich target cells are labeled.

Treatment Methods

Methods are provided for treating a disease or disorder that istreatable with an ADCC-based antibody therapy in an individual. Themethods generally involve: a) determining a category of responsivenessto an antibody therapy by genotyping the individual for an FcγRIIApolymorphism and an FcγRIIIA polymorphism; b) selecting an antibody froma set of related antibodies, where members of the set of relatedantibodies have the same antigen binding specificity, and differ inbinding affinity to an FcγRIIA and/or an FcγRIIIA receptor; and c)administering an effective amount of the antibody to the individual.

Diseases and disorders that are treatable with an ADCC-based antibodytherapy include, but are not limited to, neoplastic diseases; autoimmunediseases; allograft rejection; viral infections; bacterial infections;and parasitic infections.

Thus, in some embodiments, methods are provided for treating aneoplastic disease in an individual. The methods generally involve: a)determining a category of responsiveness to an antibody therapy for aneoplastic disease by genotyping the individual for an FcγRIIApolymorphism and an FcγRIIIA polymorphism; b) selecting an antibody froma set of related antibodies, where members of the set of relatedantibodies have the same antigen binding specificity, and differ inbinding affinity and/or in vitro ADCC activity to an FcγRIIA and/or anFcγRIIIA receptor; and c) administering an effective amount of theantibody to the individual.

The methods are useful for treating a wide variety of cancers, includingcarcinomas, sarcomas, leukemias, and lymphomas.

Carcinomas that can be treated using a subject method include, but arenot limited to, esophageal carcinoma, hepatocellular carcinoma, basalcell carcinoma (a form of skin cancer), squamous cell carcinoma (varioustissues), bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder), bronchogenic carcinoma, coloncarcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,including small cell carcinoma and non-small cell carcinoma of the lung,adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renalcell carcinoma, ductal carcinoma in situ or bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcarcinoma, uterine carcinoma, testicular carcinoma, osteogeniccarcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, etc.

Sarcomas that can be treated using a subject method include, but are notlimited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using a subject method include,but are not limited to, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Leukemias that can be treated using a subject method include, but arenot limited to, a) chronic myeloproliferative syndromes (neoplasticdisorders of multipotential hematopoietic stem cells); b) acutemyelogenous leukemias (neoplastic transformation of a multipotentialhematopoietic stem cell or a hematopoietic cell of restricted lineagepotential; c) chronic lymphocytic leukemias (CLL; clonal proliferationof immunologically immature and functionally incompetent smalllymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia,and hairy cell leukemia; and d) acute lymphoblastic leukemias(characterized by accumulation of lymphoblasts). Lymphomas that can betreated using a subject method include, but are not limited to, B-celllymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin'slymphoma; and the like.

In other embodiments, methods are provided for treating an autoimmunedisease in an individual. The methods generally involve: a) determininga category of responsiveness to an antibody therapy for an autoimmunedisease by genotyping the individual for an FcγRIIA polymorphism and anFcγRIIIA polymorphism; b) selecting an antibody from a set of relatedantibodies, where members of the set of related antibodies have the sameantigen binding specificity, and differ in binding affinity and/or invitro ADCC activity to an FcγRIIA and/or an FcγRIIIA receptor; and c)administering an effective amount of the antibody to the individual.

Autoimmune disorders include autoimmune hemolytic anemia,antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,glomerulonephritis, Crohn's Disease, Goodpasture's Syndrome, Graves'Disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmia,bullous pemphigoid, pemphigus, acute disseminated encephalomyelitis,polyendocrinopathies, purpura, Reiter's Disease, stiff-Man syndrome,inflammation, Guillain-Barre Syndrome, insulin dependent diabetesmellitus (also referred to as Type 1 diabetes), rheumatoid arthritis,autoimmune inflammatory eye disease, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, immunethrombocytopenic purpura (ITP), Sjogren's syndrome, Waldenstrom'smacroglobulinemia, encephalitis, uveitis, leukocyte adhesion deficiency,psoriatic arthritis, progressive systemic sclerosis, primary biliarycirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, systemic lupuserythematosus, polymyositis, sarcoidosis, granulomatosis, Wegener'sGranulomatosis (vasculitis), Type-II mixed cryoglobulinemia, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, Hashimoto's thyroiditis, habitual spontaneous abortions,Reynard's syndrome, glomerulonephritis, dermatomyositis, chronic activehepatitis, celiac disease, tissue specific autoimmunity, degenerativeautoimmunity delayed hypersensitivities, autoimmune complications ofacquired immunodeficiency syndrome (AIDS), atrophic gastritis,ankylosing spondylitis and Addison's disease.

In other embodiments, methods are provided for treating allograftrejection in an individual. The methods generally involve: a)determining a category of responsiveness to an antibody therapy forallograft rejection by genotyping the individual for an FcγRIIApolymorphism and an FcγRIIIA polymorphism; b) selecting an antibody froma set of related antibodies, where members of the set of relatedantibodies have the same antigen binding specificity, and differ inbinding affinity and/or in vitro ADCC activity to an FcγRIIA and/or anFcγRIIIA receptor; and c) administering an effective amount of theantibody to the individual.

In other embodiments, methods are provided for treating a viralinfection in an individual. The methods generally involve: a)determining a category of responsiveness to an antibody therapy for aviral infection by genotyping the individual for an FcγRIIA polymorphismand an FcγRIIIA polymorphism; b) selecting an antibody from a set ofrelated antibodies, where members of the set of related antibodies havethe same antigen binding specificity, and differ in binding affinityand/or in vitro ADCC activity to an FcγRIIA and/or an FcγRIIIA receptor;and c) administering an effective amount of the antibody to theindividual.

As discussed above, the presence of a particular Fcγ receptorpolymorphism predicts an individual's degree of responsiveness to anantibody therapy. Based on the individual's Fcγ receptor genotype, anantibody is chosen from a set of related antibodies. The set of relatedantibodies comprises members that have the same antigen bindingspecificity, and differ in binding affinity and/or in vitro ADCCactivity to Fcγ receptors. Where the degree of responsiveness ispredicted to be intermediate or low, an antibody is selected forenhanced binding to a given Fcγ receptor.

In some embodiments, the genotyping step identifies an FcγRIIA H/H¹³¹genotype and an FcγRIIIA V/V¹⁵⁸ genotype, and the Fc variant antibody isselected for enhanced binding and/or in vitro ADCC function to at leastone of an FcγRIIA comprising His/His¹³¹ allele and an FcγRIIIAcomprising Val/Val¹⁵⁸ allele. In other embodiments, the genotyping stepidentifies: a) a H/H¹³¹ genotype and a V/F¹⁵⁸ genotype, and wherein theFc variant antibody is selected for enhanced binding and/or in vitroADCC function to at least one of an FcγRIIA comprising H/H¹³¹ allele andan FcγRIIIA comprising V/F¹⁵⁸ allele, and b) a H/H¹³¹ genotype and aF/F¹⁵⁸ genotype, and wherein the Fc variant antibody is selected forenhanced binding and/or in vitro ADCC function to at least one of anFcγRIIA comprising H/H¹³¹ allele and an FcγRIIIA comprising F/F¹⁵⁸allele.

In other embodiments, the genotyping step identifies one of:

a) a V/F¹⁵⁸ genotype and a H/R¹³¹ genotype, and wherein the Fc variantantibody is selected for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising H/R¹³¹ allele and an FcγRIIIAcomprising V/F¹⁵⁸ allele;

b) a V/F¹⁵⁸ genotype and a R/R¹³¹ genotype, and wherein the Fc variantantibody is selected for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising R/R¹³¹ allele and an FcγRIIIAcomprising V/F¹⁵⁸ allele;

c) a F/F¹⁵⁸ genotype and a H/R¹³¹ genotype, and wherein the Fc variantantibody is selected for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising H/R¹³¹ allele and an FcγRIIIAcomprising F/F¹⁵⁸ allele;

d) a F/F¹⁵⁸ genotype and a R/R¹³¹ genotype, and wherein the Fc variantantibody is selected for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising R/R¹³¹ and an FcγRIIIAcomprising F/F¹⁵⁸ allele;

e) a V/V¹⁵⁸ genotype and a H/R¹³¹ genotype, and wherein the Fc variantantibody is selected for enhanced binding and/or in vitro ADCC functionto at least one of an FcγRIIA comprising H/R¹³¹ allele and an FcγRIIIAcomprising V/V¹⁵⁸ allele; or f) a V/V¹⁵⁸ genotype and a R/R¹³¹ genotype,and wherein the Fc variant antibody is selected for enhanced bindingand/or in vitro ADCC function to at least one of an FcγRIIA comprisingR/R¹³¹ allele and an FcγRIIIA comprising V/V¹⁵⁸ allele.

Routes of administration, formulations, as well as dosages, oftherapeutic antibodies are well known to those skilled in the art.

A therapeutic antibody is in some embodiments formulated into apreparation suitable for injection (e.g., subcutaneous, intravenous,intramuscular, intradermal, transdermal, intratumoral, peritumoral,intrathecal, or other injection routes) by dissolving, suspending oremulsifying the antibody in an aqueous solvent (e.g., saline, and thelike) or a nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

A therapeutic antibody is formulated with one or more pharmaceuticallyacceptable excipients. A wide variety of pharmaceutically acceptableexcipients are known in the art and need not be discussed in detailherein. Pharmaceutically acceptable excipients have been amply describedin a variety of publications, including, for example, A. Gennaro,“Remington: The Science and Practice of Pharmacy,” 20th edition,Lippincott, Williams, & Wilkins (2000); Pharmaceutical Dosage Forms andDrug Delivery Systems, H. C. Ansel et al., eds., 7^(th) Ed., Lippincott,Williams, & Wilkins (1999); and Handbook of Pharmaceutical Excipients,A. H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc. (2000).

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

A therapeutic antibody can be administered as an injectable formulation.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or the active ingredient encapsulated in liposomevehicles.

In some embodiments, a therapeutic antibody is delivered by bolusinjection. In other embodiments, a therapeutic antibody is delivered bya continuous delivery system. The term “continuous delivery system” isused interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery devices (e.g., pumps)in combination with catheters, injection devices, and the like, a widevariety of which are known in the art. In other embodiments, atherapeutic antibody is administered by intravenous infusion.

In some embodiments, a therapeutic antibody is administered in an amountof from about 10 mg to about 1000 mg per dose, e.g., from about 10 mg toabout 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100mg, from about 100 mg to about 125 mg, from about 125 mg to about 150mg, from about 150 mg to about 175 mg, from about 175 mg to about 200mg, from about 200 mg to about 225 mg, from about 225 mg to about 250mg, from about 250 mg to about 300 mg, from about 300 mg to about 350mg, from about 350 mg to about 400 mg, from about 400 mg to about 450mg, from about 450 mg to about 500 mg, from about 500 mg to about 750mg, or from about 750 mg to about 1000 mg per dose.

In some embodiments, the dose of a therapeutic antibody is aweight-based dose, e.g., from about 50 mg/m² to about 100 mg/m², fromabout 100 mg/m² to about 150 mg/m², from about 150 mg/m² to about 200mg/m², from about 200 mg/m² to about 250 mg/m², from about 250 mg/m² toabout 300 mg/m², from about 300 mg/m² to about 350 mg/m², from about 350mg/m² to about 400 mg/m², or from about 400 mg/m² to about 500 mg/m².

In some embodiments, multiple doses are administered. For example, insome embodiments, a therapeutic antibody is administered once per month,twice per month, three times per month, every other week (qow), once perweek (qw), twice per week (biw), three times per week (tiw), four timesper week, five times per week, six times per week, every other day(qod), daily (qd), twice a day (qid), or three times a day (tid).

In some embodiments, a therapeutic antibody is administered over aperiod of time ranging from about one day to about one week, from abouttwo weeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more.

Therapeutic formulations of an antibody may be prepared for storage aslyophilized formulations or aqueous solutions by mixing an antibodyhaving the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),i.e., buffering agents, stabilizing agents, preservatives, isotonifiers,non-ionic detergents, antioxidants, and other miscellaneous additives.See, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, Ed.(1980). Such additives must be nontoxic to the recipients at the dosagesand concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agentsinclude both organic and inorganic acids and salts thereof such ascitrate buffers (e.g., monosodium citrate-disodium citrate mixture,citric acid-trisodium citrate mixture, citric acid-monosodium citratemixture, etc.), succinate buffers (e.g., succinic acid-monosodiumsuccinate mixture, succinic acid-sodium hydroxide mixture, succinicacid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaricacid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture,tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g.,fumaric acid-monosodium fumarate mixture, etc.), fumarate buffers (e.g.,fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers (e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additionally, there may be mentioned phosphate buffers, histidinebuffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives includephenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g.,chloride, bromide, iodide), hexamethonium chloride, and alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cyclohexanol,and 3-pentanol.

“Stabilizers” may be added to ensure isotonicity of liquid compositionsof antibodies and include polyhydric sugar alcohols, preferablytrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broadcategory of excipients which can range in function from a bulking agentto an additive which solubilizes the therapeutic agent or helps toprevent denaturation or adherence to the container wall. Typicalstabilizers can be polyhydric sugar alcohols (enumerated above); aminoacids such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamicacid, threonine, etc., organic sugars or sugar alcohols, such aslactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,myoinisitol, galactitol, glycerol and the like, including cyclitols suchas inositol; polyethylene glycol; amino acid polymers; sulfur containingreducing agents, such as urea, glutathione, thioctic acid, sodiumthioglycolate, thioglycerol, alpha.-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteinssuch as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidonemonosaccharides, such as xylose, mannose, fructose, glucose;disaccharides such as lactose, maltose, sucrose and trisaccacharidessuch as raffinose; and polysaccharides such as dextran. Stabilizers maybe present in the range from 0.1 to 10,000 weights per part of weightactive protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protecta therapeutic antibody against agitation-induced aggregation, which alsopermits the formulation to be exposed to shear surface stressed withoutcausing denaturation of the protein. Suitable non-ionic surfactantsinclude polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.),Pluronic™ polyols, and polyoxyethylene sorbitan monoethers (TWEEN-20®,TWEEN-80®, etc.). Non-ionic surfactants may be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml toabout 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation may alsocontain more than one active compound as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended. The active ingredients may also be entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin micropheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Osal, Ed. (1980).

Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing anantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradable lactic acid-glycolic acid copolymers such as theLUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated antibodies remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—-S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Reagents, Devices and Kits

Also provided are reagents, devices and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devicesand kits thereof may vary greatly. Reagents and devices of interestinclude those mentioned above with respect to the methods of identifyingthe presence of the target polymorphisms, where such reagents mayinclude nucleic acid primers, arrays of nucleic acid probes, antibodiesto polymorphic polypeptides (e.g., immobilized on a substrate), signalproducing system reagents, etc., depending on the particular detectionprotocol to be performed.

In some embodiments, a subject kit comprises: i) an element forgenotyping a sample to identify an FcγRIIA polymorphism; ii) an elementfor genotyping a sample to identify an FcγRIIIA polymorphism; and iii) areference that correlates a genotype with predicted response to atherapeutic antibody. In some embodiments, the reference is a chart ortable that correlates predicted degrees of responsiveness to a giventherapeutic antibody to Fey receptor polymorphisms. Elements forgenotyping include, e.g., nucleic acid probes, and nucleic acid primersets. A sample will in some embodiments be a biological sample obtainedfrom an individual, e.g., a blood sample or other sample that includesnucleic acid (e.g., genomic DNA) from the individual.

In some embodiments, the reference indicates a high degree ofresponsiveness to a given therapeutic antibody. In these embodiments,therapeutic antibody may be selected for administration to theindividual. In other embodiments, the reference indicates anintermediate or low responsiveness; and choosing an Fc variant antibodythat exhibits enhanced binding and/or in vitro ADCC function to anFcγRIIA and/or an FcγRIIIA is indicated. In some embodiments, a subjectkit includes a set of related antibodies.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Polymorphisms in FcγRIIA and FcγRIIIA; andResponse to Rituximab

I. Materials and Methods

A. Patient Population

This study included 87 patients with follicular lymphoma, who weretreated with rituximab at Stanford Medical Center between 1993 and 2003.They were selected because of the availability of their lymphoma tumorcells, peripheral blood or serum samples, and their known clinicalresponse to rituximab. The pathology of all patient cases was reviewed.There were 47 patients with follicular small cleaved, 35 patients withfollicular mixed, and five patients with follicular large-cell lymphoma.Fifteen patients had received rituximab as their first-line therapy.Seventy-two patients had received chemotherapy before rituximab,including 10 patients who had prior bone marrow transplantation. Nopatients received chemotherapy within the 2 months before rituximabtreatment. Eighty-one patients had four weekly infusions of rituximab at375 mg/m², five patients had eight weekly infusions of 375 mg/m², andone patient had four weekly infusions of 250 mg/m². Clinical responseswere determined by physical examination and computed tomography scansbetween 1 and 3 months after last rituximab infusion and every 3 monthsthereafter. These responses were scored according to the Cheson criteria(Cheson B D et al., 1999, “Report of an international workshop tostandardize response criteria for non-Hodgkin's lymphoma,” J. Clin.Oncol. 17:1244-1253). Maximal clinical responses were observed at 1 to 3months in all but three patients, who had partial responses at 1 to 3months and showed additional tumor shrinkage at later time points.Pretreatment tumor cells were available in 43 patients and were used forin vitro ADCC assay. FcγR polymorphisms were analyzed in all 87patients. This study was conducted according to a protocol approved bythe institutional review board of our institution, and informed consentwas obtained from all patients for the use of tissue samples and theanalysis of clinical information.

B. Tumor Cells

Suspensions of pretreatment tumor cells isolated from lymph nodes werecryopreserved in liquid nitrogen. For ADCC assay, the tumor cells werethawed and subjected to Ficoll-Paque PLUS (Amersham Pharmacia Biotech,Piscataway, N.J.) gradient centrifugation to remove dead cells. Theviability of tumor cells, determined by trypan blue dye exclusion at thetime of assay, always exceeded 90%. The percentage of tumor cells ineach sample was estimated by staining with antibodies specific for kappaor lambda light chains.

C. ADCC Assay

Lymphoma cells were labeled with chromium-51 (⁵¹Cr) by incubating 3×10⁶cells with 450 μCi of ⁵¹Cr (Amersham Pharmacia Biotech) for 2 hours at37° C. Cells were washed with RPMI-1640, and then incubated for 30minutes at 37° C. with antibodies (at 10 mg/mL) Excess antibodies wereremoved by washing with medium. Mononuclear cells were obtained byFicoll-Hypaque centrifugation of peripheral blood of a healthy donor(with FcγRIIIA V/V¹⁵⁸ genotype) and used as effector cells. 1×10⁴⁵¹Cr-labeledtarget cells were incubated for 4 hours at 37° C. with theindicated number of effector cells in 200 μL of RPMI-5 medium(RPMI-1640, 10 mmol/L HEPES, 5% heat-inactivated human AB serum, 1%L-glutamine). Fifty microliters of medium was collected after 4 hours ofincubation and counted in a MicroBeta 1450 scintillation counter(Wallac, Turku, Finland). Spontaneous ⁵¹Cr release was determined in theabsence of effector cells. Maximal ⁵¹Cr release was determined by lysiswith 0.5% Triton X-100. All samples were assayed in triplicate. Thespecific ⁵¹Cr release was determined by subtracting the spontaneous ⁵¹Crrelease from that of the treatment wells, then dividing the result bythe maximal ⁵¹Cr release minus spontaneous ⁵¹Cr release. All the tumorsamples had coexistent T-cells of variable degree (Table-1). To comparedifferent tumor samples, the specific ADCC is calculated by dividing thespecific ⁵¹Cr release in rituximab-treated samples minus ⁵¹Cr release incontrol IgG1-treated samples by the percentage of CD20-positive cells inindividual samples.

D. Analysis of FcγRIIIA and FcγRIIA Polymorphisms

Genomic DNA was prepared from tumor cells or from peripheral-bloodmononuclear cells using a DNA extraction kit (Qiagen, Valencia, Calif.).In six patients, DNA was prepared from the serum using a describedmethod (Kopreski M S, Benko F A, Kwee C, et al., 1997, “Detection ofmutant K-ras DNA in plasma or serum of patients with colorectal cancer,”Br. J. Cancer 76:1293-1299). Genotyping of FcγRIIIA V/F¹⁵⁸ and FcγRIIAH/R¹³¹ polymorphisms was performed by a polymerase chain reactionfollowed by allele-specific restriction enzyme digestion (Koene H R,Kleijer M, Algra J, et al., 1997, “Fc-gamma-RIIIA-158 V/F polymorphisminfluences the binding of IgG by natural killer cell Fc-gamma-RIIIA,independently of the Fc-gamma-RIIIA-48 L/R/H phenotype,” Blood90:1109-1114; Jiang X-M, Arepally G, Poncz M, et al., 1996, “Rapiddetection of the Fc-gamma-RIIA-H/R131 ligand-binding polymorphism usingan allele-specific restriction enzyme digestion (ASRED),” J. Immunol.Methods 199:55-59). All genotyping of FcγRIIIA polymorphism wasconfirmed by direct sequencing of the region of interest.

E. Statistical Analysis

Differences in the means of ADCC killing were tested by single-factoranalysis of variance test and checked by the Kruskal-Wallis(nonparametric) test. The clinical responses of the patients werecompared using a two-tailed Fisher's exact test (PRISM for Macintosh;GraphPad Software, San Diego, Calif.). A logistic regression analysisincluding age (.gtoreq. or <60 years), stage (III.nu. IV), presence ofbulky disease, number of extranodal sites (.gtoreq.two or <two), priorbone marrow transplantation, and FcγRIIA and FcγRIIIA genotype was usedto identify independent prognostic variables influencing the clinicalresponses (StatView 5.0.1; SAS Inc, Cary, N.C.).

II. Results

A. Rituximab-Mediated ADCC in Follicular Lymphoma Cells

The ability of rituximab to mediate ADCC in follicular lymphoma cellswas determined. Pretreatment lymphoma cells from 43 patients were testedusing effector cells isolated from one healthy donor. Rituximab-mediatedADCC was detected in all 43 patient samples (range, 13.5% to 100%). Asexpected, the murine antibody of rituximab, 2B8, which contains a mouseβ1 Fc portion and binds lymphoma cells identically to rituximab, did notmediate ADCC (data not shown).

The relation of the observed ADCC susceptibility of lymphoma cells fromindividual patients to their clinical response to rituximab therapy wasthen evaluated. Patients were subdivided into nonresponders (NR),partial responders (PR), and complete responders (CR) according to theirresponse to rituximab at the first evaluation at 1 to 3 months (Table 1,FIG. 5). The range of ADCC varied widely in all three Groups (NR, 16.9%to 80.6%; PR, 13.5% to 57.0%; CR, 20.9% to 100.0%; FIG. 1). However,there was no difference of rituximab-mediated ADCC between the three

Groups (means.+−.standard deviations: NR, 44.6%.+−.18%; PR,40.0%.+−.12%; CR, 53.6%.+−.23%). Additional analysis showed norelationship between rituximab-mediated ADCC and response when clinicalresponse was scored at 6, 9, or 12 months after treatment, nor did thesusceptibility to ADCC correlate with the duration of remission (datanot shown). In a subGroup of 29 patients whose tumors were studied in areport on complement-mediated cytotoxicity, (Weng W- K, Levy R, 2001,“Expression of complement inhibitors CD46, CD55, and CD59 on tumor cellsdoes not predict clinical outcome after rituximab treatment infollicular non-Hodgkin lymphoma,” Blood 98:1352-1357) the expression ofCD20 on their lymphoma cells had previously been determined by flowcytometric staining. Within this subGroup, there was no correlationbetween the expression of CD20 and rituximab-mediated ADCC (r=−0.03;P=0.88).

B. Clinical Response to Rituximab Therapy and FcγRIIIA V/F¹⁵⁸Polymorphism

The FcγRIIIA (CD16) of V allele demonstrates higher affinity to IgG1than the F allele and mediates ADCC more effectively. Recently, Cartronet al., (Cartron G, Dacheux L, Salles G, et al., 2002, “Therapeuticactivity of humanized anti-CD20 monoclonal antibody and polymorphism inIgG Fc receptor Fc-gamma-R IIIA gene,” Blood 99:754-758) have shown anassociation between FcγRIIIA V/V¹⁵⁸ genotype and higher response rate inpatients treated with first-line rituximab. This association was assayedin the subject patient Group, the majority of whom were treated forrelapsed disease. The study Group was expanded to 87 by acquiringperipheral blood or serum samples from additional rituximab-treatedpatients.

In this sample set, 13 patients (15%) had homozygous VN (V/V¹⁵⁸), 40(46%) had heterozygous V/F (V/F¹⁵⁸), and 34 (39%) had homozygous F/F(F/F¹⁵⁸). The three groups were not different in terms of average age atthe time of treatment, number of prior chemotherapy courses, or timebetween diagnosis and treatment (Table 2, FIG. 6). The response rate inpatients with V/F¹⁵⁸ and in patients with F/F¹⁵⁸ was similar at all fourtime points (Table 3, FIG. 7). For that reason, we Grouped V/F¹⁵⁸ andF/F¹⁵⁸ together as the F carrier for statistical analysis. A significantdifference was detected between the response rates of V/V¹⁵⁸ and Fcarriers (Table 3, FIG. 7). The progression-free survival (PFS) at 2years was 45% for patients with 158 VN, 12% for 158 V/F, 16% for 158F/F, and 14% for F carriers, using the Kaplan-Meier estimation, withmedian time to progression (TTP) of 534, 148, 250, and 170 days for eachGroup, respectively. The PFS estimate of patients with V/V¹⁵⁸ wassignificantly longer than that for patients with V/F¹⁵⁸, F/F¹⁵⁸, or Fcarriers (FIG. 2).

C. Clinical Response to Rituximab Therapy and FcγRIIA H/R¹³¹Polymorphism

The FcγRIIA (CD32) is another activating FcγR that is expressed only onmacrophages but not on natural killer (NK) cells. An H/R polymorphism atposition 131 of FcγRIIA has been found to affect its affinity to humanIgG (Jiang X- M, Arepally G, Poncz M, et al., 1996, “Rapid detection ofthe Fc-gamma-RIIA-H/R131 ligand-binding polymorphism using anallele-specific restriction enzyme digestion (ASRED),” J. Immunol.Methods 199:55-59). Of the 87 patients in the Group, 20 (23%) hadhomozygous H/H(H/H¹³¹), 43 (49%) had heterozygous H/R(H/R¹³¹), and 24(28%) had homozygous R/R(R/R¹³¹). Once again, the three Groups were notdifferent in terms of average age at the time of treatment, number ofprior chemotherapy treatments, or time between diagnosis and treatment(Table 2). Although there was no difference in the response rate at 1 to3 months between the three Groups, patients with H/H¹³¹ showed asignificantly higher response rate than the other two Groups combined(H/R and R/R (R carrier)) at 6, 9, and 12 months (Table 4, FIG. 8). Thishigher response rate also translated to longer remission: the PFS at 2years was 37% for patients with H/H¹³¹, 13% for H/R¹³¹, 19% for R/R¹³¹,and 14% for R carrier using the Kaplan-Meier estimation with TTP of 445,162, 158, and 158 days for each Group, respectively. The PFS estimatefor patients with H/H¹³¹ was significantly longer than for patients withother genotypes (FIG. 3).

The possibility of an association between FcγRIIIA and FcγRIIA genotypesthat might explain the correlation of the two with response rate wasexamined. As shown in Table 5, FIG. 9, there was no significantdifference in the fraction of V/V¹⁵⁸ or F carrier in three H/R¹³¹genotypes. The combination of FcγRIIIA V/V¹⁵⁸ and/or FcγRIIA H/H¹³¹ wasthen analyzed, and their relationship to rituximab response. As shown inTable 6, FIG. 10, patients with V/V¹⁵⁸ and/or H/H¹³¹ (total of 30patients) had a significantly higher response rate than patients withouteither genotype at all four time points (83% .nu. 54%, P=0.009 at 1 to 3months; 80% .nu. 34%, P=0.0001 at 6 months; 69% .nu. 26%, P=0.0003 at 9months; 59% .nu. 18%, P=0.0004 at 12 months). The PFS estimate ofpatients with V/V¹⁵⁸ and/or H/H¹³¹ was also significantly longer(P=0.001), with TTP of 445 and 140 days for the two Groups, respectively(FIG. 4). By logistic regression analysis, FcγRIIIA V/V¹⁵⁸ genotypeemerged as the only predictive factor for response at 1 to 3 months,whereas both the FcγRIIIA V/V¹⁵⁸ genotype and FcγRIIA H/H¹³¹ genotypewere identified as independent predictive factors for response at 6, 9,and 12 months (Table 7, FIG. 11).

III. Discussion

In this study, the observation of Cartron et al., supra, that V/V¹⁵⁸genotype is associated with higher response rate to rituximab treatmentwas confirmed. However, there were significant differences between thepresent and prior studies. First, the response rate in the patient Groupof the present study was lower than that in the previous report,especially at 12 months after treatment. This observation is consistentwith previous observations of a lower response rate when rituximab isused as second-line treatment. In addition, the patients of the presentstudy probably had higher tumor burden because 53% of them had bulky (5cm) disease compared with the previous study in patients with nonbulkydisease. Second, although F carriers (V/F and F/F) showed asignificantly lower response rate, the response rate in patients withF/F¹⁵⁸ was slightly higher than that in patients with V/F¹⁵⁸. Thebiologic explanation of this phenomenon is unclear, given that patientswith V/F¹⁵⁸ would be expected to have an intermediate response rate.Third, consistent with the previous report, we detected a differencebetween V/V¹⁵⁸ and F carrier. However, one interesting observation inthis study is that the difference became more pronounced after longertimes from the treatment. The antibody is known to persist for up to 6months, and its effect may be cumulative.

The most unexpected result came from the analysis of FcγRIIApolymorphism. The allele of H/H¹³¹ binds to human IgG2 better than thatof R/R¹³¹. However, no significant difference in the affinity of thesetwo allelic forms for human IgG1 has been noted. (Parren P W, WarmerdamP A, Boeije L C, et al., 1992, “On the interaction of IgG subclasseswith the low affinity Fc-gamma-RIIA (CD32) on human monocytes,neutrophils, and platelets: Analysis of a functional polymorphism tohuman IgG2,” J. Clin. Invest. 90:1537-1546). Therefore, it wasunexpected to find a higher rituximab response rate associated withH/H¹³¹ genotype (Table 4, FIG. 8). Similar to the FcγRIIIA V/F¹⁵⁸polymorphism, a gene dosage effect of the 131H allele was not observed.Instead, the response rate in patients with H/R¹³¹ was similar to thatof R/R¹³¹ at 6, 9, and 12 months. The biologic explanation of thisobservation is not clear. The association between FcγRIIA H/H¹³¹ andhigher response rate was not a result of a linkage disequilibrium ofFcγRIIIA V/F¹⁵⁸ polymorphism (Table 5, FIG. 9). There is a randomdistribution of combinations of variant genotypes of FcγRIIA andFcγRIIIA in the normal population. (Lehrnbecher T, Foster C B, Zhu S, etal., 1999, “Variant genotypes of the low-affinity Fc-gamma receptor intwo control populations and a review of low-affinity Fc-gamma receptorpolymorphisms in control and disease populations,” Blood 94:4220-4232).

The FcγRIIA H/R¹³¹ polymorphism is an independent predictive factor forclinical response: In the subGroup of patients with 158 F carrier,FcγRIIA H/H¹³¹ genotype was associated with higher response rate at 6,9, and 12 months (H/H=76% .nu. R carrier=34%, P=0.004 at 6 months;H/H=65% .nu. R carrier=26%, P=0.007 at 9 months; H/H=47% .nu. Rcarrier=18%, P=0.026 at 12 months). Furthermore, all three patients withboth V/V¹⁵⁸ and H/H¹³¹ genotypes had long-lasting remissions (Table 6,FIG. 10). Patients with V/V¹⁵⁸ and/or H/H¹³¹ genotypes showed a higherresponse rate and a longer remission than did patients without either ofthese two genotypes (Table 6, FIG. 10). Lastly, the logistic regressionanalysis showed that the V/V¹⁵⁸ and H/H¹³¹ were independent predictivefactors for response at 6, 9, and 12 months. The report of Cartron etal., supra, also analyzed the FcγRIIA H/R¹³¹ polymorphism and concludedthat the FcγRIIA polymorphism did not influence the clinical response.However, it is important to point out that Cartron et al., analyzed asmaller Group of patients (N=45) and scored the clinical responses onlyat 1 and 12 months. In this study, the most prominent differences wereobserved at 6 and 9 months (Table 4, FIG. 8).

To reiterate, this study established a) for the first time a clear rolefor ADCC in the clinical effects (treatment response and freedom ofprogression) of rituximab at the level of the effector cell wasestablished through in vitro ADCC assays and by correlating this tospecific FcγRIIIA polymorphism, b) through a rigorous time-courseanalysis, an unequivocal proof that FcγRIIA H/R¹³¹ polymorphism isindependently associated with the therapeutic response rate, and c) boththe FcγRIIIA V/F¹⁵⁸ and the FcγRIIA H/R¹³¹ polymorphisms areindependently and collectively associated with the therapeutic responserate and freedom from progression, which is to say that details of bothpolymorphisms are absolutely essential to make a meaningful predictionof the therapeutic response rate. This work has provided a sequentialcorrelation between FcγRIIIA V/F¹⁵⁸ polymorphism, in vitro ADCC, and theclinical effect of rituximab. Although similar in vitro ADCC experimentswere not conducted to test such a relationship for FcγRIIA H/R¹³¹polymorphism in these patients, the direct correlation between theH/R¹³¹ polymorphism and the clinical effect of rituximab isunequivocally established in this study. Thus, given the central rolefor these two receptors in ADCC, a) patients can be classified into nineGroups based on these polymorphisms, b) it is possible to generate Fcvariant antibodies specific for FcγRIIIA and FcγRIIA alleles byselecting for enhanced binding and/or in vitro ADCC function, c) it iscomprehended that, like rituximab, many other antibodies exert theirtherapeutic efficacy through ADCC as the major mechanism of action, andtherefore, d) the patient Group specific Fc variants contemplated inthis disclosure can be used to enhance the therapeutic response of theseantibodies to treat ADCC-treatable diseases or disorders.

Example 2 Construction of Patient Group Specific Fc Variant Antibodies

Throughout this example, methods are described to generate Fc variantsoptimized for FcγRIIIA F/F¹⁵⁸ and FcγRIIA R/R¹³¹ genotypes (PatientGroup-IX; See, e.g., Table D). While Rituximab is shown as an example,any other ADCC-dependent antibody can be used in these experiments withappropriate modifications in the procedures. For preliminary bindingstudies which involve SSM and Fc Walking, aglycosylated Fc fragmentincluding the hinge as well as the soluble domains of FcγRIIIA andFcγRIIA are expressed in vitro or through bacterial expression byestablished procedures (Kim et al., 1994, Eur. J. Immunol. 24:542;Sondermann and Jacob, 1999, Biol. Chem. 380:717-721).

FC ENGINEERING AND BINDING STUDIES: For each chosen residue, in vitroscanning saturation mutagenesis (SSM) is carried out (U.S. Pat. No.6,180,341). Briefly, at each site, twenty-one genes encoding allpossible amino acid substitutions as well as a double stop codon(control) are constructed by overlap extension PCR. The followingconserved stretches of the Fc region will be subjected to both SSM andFc Walking, one residue at a time, in order to generate single mutants:L²³⁴-S²³⁹ (6×20=120 variants), R²⁵⁵-T²⁶⁰ (6×20=120 variants); D²⁶⁵-E²⁶⁹(5×20=100 variants); N²⁹⁷-T²⁹⁹ (3×20=60 variants); A³²⁷-I³³² (6×20=120variants) (See, e.g., FIG. 12-C). In addition, approximately 5-10residues upstream and downstream of the conservative stretches will alsobe subjected to Fc Walking (FIG. 12-D). Taken together, this generates<2000 Fc variants. Similarly, double and triple mutants are generated bycombining the single mutants and additionally selecting for the bindingproperties (Yang et al., 1995, J. Mol. Biol. 254:392; Wu et al., 1998,Proc Natl. Acad. Sci. USA 95:6037). This is accomplished by simultaneousSSM at 3-5 different positions. The final products of the overlapextension PCR reaction contain a T7 promoter and ribosome binding sitein front of the Fc fragment gene. An HSV sequence is also present at theC-terminal end of the Fc fragment gene, so that the MAb fragment proteincan be detected by ELISA using an anti-HSV monoclonal antibody. The PCRoverlap extension products are used as templates for coupled in vitrotranscription-translation reactions to produce Fc variants. An E. coliS30 ribosomal extract is used for in vitro translation.

The protein products from the coupled in vitro transcription-translationstep are analyzed by ELISA. In ELISA, 96-well microtiter plates arecoated with the BSA conjugate of soluble FcγRIIIA F¹⁵⁸ or FcγRIIA R¹³¹.The plates are then incubated with equal amounts from each of the invitro synthesis reactions. In order to provide accurate calibration, theconstruct prepared with the Fc wild-type sequence is used on each ELISAplate. The Fc wild-type construct is produced by the overlapping PCRmethod alongside the mutants, thereby providing an accurate calibrationfor all stages of the procedure. Heterogeneity of thetranscribed/translated products is verified by DNA/protein sequencingprotocols, or matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF-MS). The ELISA results for the differentmutants are recorded and the promising Fc variants in terms of bindingand affinity are subjected to in vitro ADCC assays (See, e.g., below).

Construction, Expression, and Purification of Fc Variant Antibodies andFcγRs:

The wild-type Fc region of rituximab is substituted with Fc variantsgenerated through Fc engineering, cloned and expressed in 293T cells,and purified by using protein-A chromatography. FcγRs are constructed asC-terminal 6×His-GST fusions, expressed in 293T (human FcγRs) or NIH 3T3(mouse FcγRIII) cells, and purified by using nickel affinitychromatography. Heterogeneity of the translated products is tested byany one of the following analytical procedures: SDS-PAGE, gelfiltration, MALDI-TOF-MS.

Binding Assays: AlphaScreen assays use untagged Ab to compete theinteraction between biotinylated IgG bound to streptavidin donor beadsand FcγR-His-GST bound to anti-GST acceptor beads. Competition SPR(Harvey et al., 2004, Proc. Natl. Acad. Sci. USA 101:9193) experimentsmeasured capture of free Ab from a preequilibrated Ab:receptor analytemixture to FcγRIIIA V¹⁵⁸-His-GST bound to an immobilized anti-GSTsurface. Equilibrium dissociation constants (K_(D) values) arecalculated by using the proportionality of initial binding rate on freeAb concentration in the Ab:receptor equilibrium (Schier et al., 1996, J.Mol. Biol. 255:28; Daugherty et al., 1998, Protein Engg. 11:825-832).

in vitro ADCC is measured by ⁵¹Cr release assay (Weng and Levy, 2003, J.Clin. Oncol. 21:3940). Human PBMCs are purified from leukopacks by usinga Ficoll gradient and allotyped for FcγRIIIA F/F¹⁵⁸ and FcγRIIA R/R¹³¹by PCR. NK cells are isolated from human PBMCs by negative selectionusing a magnetic bead NK cell isolation kit (Miltenyi Biotech, Auburn,Calif.). All the necessary target cell lines are obtained from AmericanType Culture Collection. As a second format, ADCC assay using PBMC aseffector cells is measured based on lactate dehydrogenase activityreleased from the dead or plasma membrane damaged cells (Shields et al.,2001, J. Biol. Chem. 276:6591).

For phagocytosis experiments, monocytes are isolated from PBMCs ofindividuals belonging to the Patient Group-IX (FcγRIIIA F/F¹⁵⁸ andFcγRIIA R/R¹³¹) by using a Percoll gradient and differentiated intomacrophages by culturing in a medium supplemented with 0.1 ng/mlgranulocyte macrophage colony-stimulating factor for 1 week. Forimaging, WIL2-S target cells are labeled with PKH67 (Sigma) andcocultured for 24 h with macrophages at an effector:target cell ratio of3:1 in the presence of 100 ng/ml Fc variant rituximab optimized for thePatient Group-IX. Cells are then treated with secondary antibodiesanti-CD11-RPE and anti-CD14-RPE (DAKO) for 15 minutes before live cellimaging using a fluorescence microscope. For quantitative ADCP(antibody-dependent cell-mediated phagocytosis), WIL2-S target cells arelabeled with PKH67, seeded in a 96-well plate at 20×10³ cells per well,and treated with WT or the Fc variant rituximab at the designated finalconcentrations. Macrophages are labeled with PKH26 (Sigma) and added tothe opsonized labeled target cells at 20×10³ cells per well, and thecells are cocultured for 18 h. Fluorescence is measured by usingdual-label flow cytometry. Fc variant antibodies with increased potencyand efficacy are selected. CDC assays are performed according topublished procedures (Gazzano-Santoro et al., 1997, J. Immunol. Meth.202:163; Uchida et al., 2004, J. Exp. Med. 199:1659).

In Vivo B-Cell Depletion: Cynomolgus monkeys (Macaca fascicularis) willbe injected intravenous once daily for 4 consecutive days with wild-typeor the Fc variant rituximab optimized for the Patient Group-IX (Reff etal., 1994, Blood 83:435-445). The test animals will be allotyped for theFcγR polymorphisms, and if required, the animals corresponding to F/F¹⁵⁸and R/R¹³¹ genotypes will be used for the experiment. The experiment iscomprised of six treatment Groups of .about.0.1, 0.2, 2, 7, or 30 μg/kg(Group-IX specific variant rituximab) or .about.2 or 30 μg/kg (wild-typecontrol), with three monkeys per treatment Group. Blood samples areacquired on two separate days before dosing (baseline) and at days 1, 2,5, 15, and 28 after initiation of dosing. For each sample, cellpopulations are quantified by flow cytometry by using specificantibodies against the representative marker antigens. Percent B-celldepletion is calculated by comparing B-cell counts on the given day withthe average of the two baseline measures for each animal.

Example 3 ADCC Improvement Through CDR Engineering

Antibodies or antibody fragments are specifically optimized to a patientGroup according to their FcγRIIA and FcγRIIIA polymorphism as describedin Example 2. However, instead of introducing amino acid modifications(i.e., insertions, substitutions, deletions, etc.) into the Fc portionof the antibody, as described in Example 2, modifications are introducedin the complementarity determining regions (CDRs) of the antibody.

CDR engineering of Rituxan is shown here as an example. FIG. 16 showsthe codon-based mutagenesis of the light chain CDR2 region (APSNLAS).Scanning saturation mutagenesis (Burks et al., 1997, Proc. Natl. Acad.Sci. USA 94:412; Chen et al., 1999, Protein Eng. 12:349) is used in astepwise fashion to rapidly improve the affinity of the CD20-bindingfragment by greater than 50-100 fold. The focused library of V_(L) CDR2region will have 140 variants. In the absence of structural informationabout the Rituxan-CD20 interactions, in vitro translated antibodylibraries for all six light and heavy chain CDRs will be screened by aquantitative assay (k_(on), k_(off), K_(d), in vitro ADCC assay) toidentify variants with improved binding to CD20. The Rituxan variants inthese libraries each contains a single mutation, and all 20 amino acidsare introduced at each CDR residue, resulting in a library consisting of1,320 unique variants. Multiple clones displaying 2- to >10 foldimproved affinity are identified. The CDR residues identified as“critical” (optimal) residues for engineering (FIG. 16) will besubjected to second round of scanning saturation mutagenesis. Wherenecessary, simultaneous mutations of 3-4 changes in multiple CDRsequences are made. Promising scFv variants were converted into IgG1format, cloned, and expressed in 293T cells, and purified by standardprocedures. Binding assays and in vitro ADCC assays are carried outessentially as described in Example II.

Example 4 Engineering, Screening and Selection of Optimized PatientGroup Variant Rituximab Antibodies

Antibodies or antibody fragments are specifically optimized for apatient Group based on their FcγRIIA and FcγRIIIA polymorphism. Forexample, patients are categorized into Groups according to their FcγRIIA(H/R¹³¹) polymorphism and their FcγRIIIA (V/F¹⁵⁸) polymorphism.Consequently, patients can be divided into nine genotypic Groups,including: V/V¹⁵⁸, H/H¹³¹ (Group-I); V/F¹⁵⁸, H/H¹³¹ (Group-II); F/F¹⁵⁸,H/H¹³¹ (Group-III); V/V¹⁵⁸, H/R¹³¹ (Group-IV); V/F¹⁵⁸, H/R¹³¹ (Group-V);F/F¹⁵⁸, H/R¹³¹ (Group-VI); V/V¹⁵⁸, R/R¹³¹ (Group-VII); V/F¹⁵⁸, R/R¹³¹(Group-VIII); and F/F¹⁵⁸, R/R¹³¹ (Group-IX) (See, e.g., Table D).Notably, an antibody can be optimized for each patient Group except forthe Group or Groups that exhibit the highest degree of responsiveness tothe antibody therapy.

A variant antibody specific for a particular patient Group (genotype) isengineered such that the variant antibody exhibits responsivenessoptimized to the patient Groups (genotypes) which exhibit a higherdegree of responsiveness to the parent antibody. In some embodiments, avariant antibody specific for a particular patient Group (genotype) isengineered such that the variant antibody exhibits responsivenessoptimized to the patient Group which exhibits the highest degree ofresponsiveness to the parent antibody.

Responsiveness to an antibody therapy for a neoplastic disease caninclude one or more of: antibody-dependent cell-mediated cytotoxicity(ADCC) response to tumor cells; reduction in tumor mass; reduction innumber of tumor cells.

Responsiveness to an antibody therapy for an autoimmune disease caninclude one or more of: reduction in a symptom associated with theautoimmune disorder; reduction in the number and/or activity of anautoreactive B-cell; reduction in the number and/or activity of anautoreactive T-cell; etc.

Responsiveness to an antibody therapy for allograft rejection caninclude one or more of: reduction in the amount of immunosuppressivedrug that must be administered to an individual who is the recipient ofan allograft and still maintain the allograft; duration of maintenanceof the allograft; function of the allograft; reduction in the numberand/or activity of alloreactive T-cells in the allograft recipient.

Responsiveness to an antibody therapy for a viral infection can includeone or more of: reduction in the number of viral genomes in a tissue,fluid, or other specimen from an individual; reduction in one or moresymptoms of a viral infection; etc.

Responsiveness may be determined at various times after treatment with agiven antibody therapy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12months following treatment, e.g., following initiation of treatment.Thus, e.g., responsiveness can be expressed with a time component.

For example, when Rituximab is the antibody of interest, a variantRituximab antibody is engineered for each patient Group, except theGroup associated with the highest degree of responsiveness to Rituximab(Group-I). Variant Rituximab antibodies are then optimized for eachpatient Group such that each Group exhibits an increased degree ofresponsiveness to the variant antibody. In some embodiments, the variantRituximab antibodies are optimized for each patient Group exhibit asimilar degree of responsiveness as exhibited by the patients of Group-Ito Rituxamab. Given that Group-I genotype is associated with the highestdegree of responsiveness to Rituxamab, variant Rituximab antibodies areonly engineered and optimized for patient Groups II-IX.

While the example described below are directed toward the V/F¹⁵⁸, H/H¹³¹genotype, it will be understood that the same protocols may be employedto engineer optimized antibodies for the F/F¹⁵⁸, H/R¹³¹ genotype(Group-III); the V/V¹⁵⁸, H/R¹³¹ genotype (Group-IV); the V/F¹⁵⁸, H/R¹³¹genotype (Group-V); the F/F¹⁵⁸, H/R¹³¹ genotype (Group-VI); the V/V¹⁵⁸,R/R¹³¹ genotype (Group-VII); the V/F¹⁵⁸, R/R¹³¹ genotype (Group-VIII);and the F/F¹⁵⁸, R/R¹³¹ genotype (Group-IX) (See, e.g., Table D).

A. Patient Group-II (V/F¹⁵⁸, H/H¹³¹ Genotype)

Variant Rituximab antibodies engineered for Group-II (V/F¹⁵⁸, H/H¹³¹genotype) are optimized to exhibit an increased degree of responsivenesscomparable to any other Group with a higher responsiveness to theantibody. In some embodiments, variant Rituximab antibodies engineeredfor Group-II (V/F¹⁵⁸, H/H¹³¹) are optimized to exhibit a degree ofresponsiveness comparable to the level of H/H¹³¹) to Rituxamab.

1. Engineering Variant Rituximab Antibodies for Patient Group-II(V/F¹⁵⁸, H/H¹³¹ Genotype)

The Fc region and CDRs of Rituximab may be engineered to create variantRituximab antibodies. Antibody variants can be obtained by substitution,deletion, inversion, and/or insertion of any of the amino acids presentin the antibody. For example, Fc variant antibodies can be obtained bysubstitution of any of the amino acids present in the Fc fragment.Likewise, CDR variant antibodies can be obtained by deletion of any ofthe amino acids present in the CDRs. As can be appreciated, there arepositions in the sequence that are more tolerant to substitutions,deletions, inversions, and/or insertions than others, and with someamino acid modifications improving the binding activity of the parentantibody. The amino acids that are essential should either be identicalto the amino acids present in the parent antibody, or substituted byconservative substitutions. The amino acids that are non-essential canbe identical to those in the parent antibody, or can be substituted byconservative or non-conservative substitutions, and/or can be deleted.

The substitutions, deletions, inversions, and/or insertions of aminoacids in an Fc variant antibody will occur in regions not essential toantigen binding. The identification of essential and non-essential aminoacids in the antibody can be achieved by methods known in the art, suchas by site-directed mutagenesis (for example, SSM) and AlaScan analysis(Moffison et. al., 2001, Chem. Biol. 5:302-307).

Essential amino acids have to be maintained or replaced by conservativesubstitutions in the variants. Non-essential amino acids can be deleted,or replaced by a spacer or by conservative or non-conservativesubstitutions.

The following conserved stretches (bolded and underlined) in the Fcregion of Rituximab (SEQ ID NO: 1) may be subjected to both SSM (See,e.g., U.S. Pat. No. 6,180,341) and Fc Walking, one residue at a time, inorder to generate single mutants: L234-S²³⁹ (6×20=120 variants),R²⁵⁵-T²⁶⁰ (6×20=120 variants); D²⁶⁵-E²⁶⁹ (5×20=100 variants); N²⁹⁷-T²⁹⁹(3×20=60 variants); A³²⁷-I³³² (6×20=120 variants) (See, e.g., FIG.12-C). Briefly, at each site, twenty-one genes encoding all possibleamino acid substitutions as well as a double stop codon (control) areconstructed by overlap extension PCR. In addition, approximately 5-10residues upstream and downstream of the lower hinge, B/C loop, C′/E loopand F/G loop are also subjected to Fc Walking (FIG. 12-D). Takentogether, this generates <2000 Fc variants.

Similarly, the Fc region in REMICADE® (SEQ ID NO: 5), ERBITUX® (SEQ IDNO: 7), HERCEPTIN® (SEQ ID NO: 9) and CAMPATH® (SEQ ID NO: 11) may besubjected to Fc walking.

For example, the first conserved stretch (from the N-terminus) in the Fcregion of RITUXAN begins with an lysine (as shown below). This arginineresidue may be altered with any of the other known 21 amino acidresidues, for instance, tyrosine. Additionally, an antibody molecule maycomprise two or more modifications in one or more conserved regions ofthe Fc.

Rituximab Heavy Chain (Fc-amino acid residues 232- 448) (SEQ ID NO: 1)PE LLGGPS VFLFPPKPKDTLMIS RTPE VTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

Similarly, double and triple mutants are generated by combining thesingle mutants and additionally selecting for binding properties (Yanget al., 1995, J. Mol. Biol. 254:392; Wu et al., 1998, Proc Natl. Acad.Sci. USA 95:6037). This is accomplished by simultaneous SSM at 3-5different positions.

The final products of the overlap extension PCR reaction contain a T7promoter and ribosome binding site in front of the antibody fragmentgene. An HSV sequence is also present at the C-terminal end of theantibody fragment gene so that the MAb fragment protein can be detectedby ELISA using an anti-HSV monoclonal antibody. The PCR overlapextension products are used as templates for coupled in vitrotranscription-translation reactions to produce antibody variants. An E.coli S30 ribosomal extract is used for in vitro translation.

Alternatively or in addition to the modification(s) made to the Fcregion of rituximab, the antibody may be modified in one or more of itscomplementarity determining regions (CDRs) by the methods describedabove for the Fc region. Specifically, one or more modifications aremade to one or more of the heavy or light chain CDRs in rituximab (SEQID NO: 2 and 3, respectively). Any one or more of the CDR sequencespresented below (bolded and underlined) from the Rituximab heavy orlight chain may be modified by the SSM procedure.

Similarly, one or more of the above-described modifications may be madeto the CDRs regions of REMICADE® (SEQ ID NO: 4 and 5), ERBITUX® (SEQ IDNO: 6 and 7), HERCEPTIN® (SEQ ID NO: 8 and 9) and CAMPATH® (SEQ ID NO:10 and 11).

Rituximab Light Chain QIVLSQSPAILSASPGEKVTMTC RASSSVSYMHVVYQQKPGSSPKPWIY APSNL AS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSFNPPTFGAG TKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNR(SEQ ID NO: 2) Rituximab Heavy Chain QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMH VVVKQTPRQGLEWIG AIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCAR VVYY SNSYWYFDVWGTGTTVTVSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)

For example, the first CDR (from the N-terminus) in the light chainmolecule begins with an arginine (as shown below). This arginine residuemay be altered with any of the other known 21 amino acid residues, forinstance, tyrosine. Additionally, an antibody molecule may comprise twoor more modifications in its CDR. When two or more amino acidmodifications are present, all may be located in the CDR of a lightchain molecule. Alternatively, all the modification may be located inthe CDR of a heavy chain molecule. Moreover, the two or moremodifications may be present in both the heavy chain molecule and thelight chain molecule.

2. Screening Variant Rituximab Antibodies Specific for Group-II (V/F¹⁵⁸,H/H¹³¹ Genotype)

Variant Rituximab antibodies or antibody fragments engineered forGroup-II are screened using any of the functional assays describedsupra. Exemplary screening methods are described below.

For example, variant antibodies or antibody fragments may be analyzed byELISA. In ELISA, 96-well microtiter plates are coated with the BSAconjugate of soluble FcγRIIIA V/F¹⁵⁸ or FcγRIIA H/H¹³¹. The plates arethen incubated with equal amounts of variant Rituximab antibodies orantibody fragments from each of the in vitro synthesis reactions.Heterogeneity of the transcribed/translated products is verified byDNA/protein sequencing protocols, or matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The ELISA results for the different mutants are recorded and thepromising variant Rituximab antibodies or antibody fragments in terms ofbinding and affinity are subjected to in vitro ADCC assays (See, e.g.,below).

Additionally, variant Rituximab antibodies or antibody fragmentsengineered for Group-II are screened for binding affinity to FcγRIIA andFcγRIIIA. One such affinity assay, AlphaScreen, uses untagged antibodyto compete the interaction between biotinylated IgG bound tostreptavidin donor beads and FcγR-His-GST bound to anti-GST acceptorbeads. Competition SPR (Harvey et al., 2004, Proc. Natl. Acad. Sci. USA101:9193) experiments measured capture of free antibody from apre-equilibrated Ab:receptor analyte mixture to FcγRIIIA V¹⁵⁸-His-GSTbound to an immobilized anti-GST surface. Equilibrium dissociationconstants (K_(D) values) are then calculated by using theproportionality of initial binding rate on free antibody concentrationin the Ab:receptor equilibrium (Schier et al., 1996, J. Mol. Biol.255:28; Daugherty et al., 1998, Protein Eng. 11:825-832).

Further, variant Rituximab antibodies or antibody fragments may bescreened for ADCC activity. To assess ADCC activity an antibody ofinterest is added to target cells in combination with immune effectorcells, which may be activated by the antigen antibody complexesresulting in cytolysis of the target cell. Cytolysis is generallydetected by the release of label (e.g., radioactive substrates,fluorescent dyes or natural intracellular proteins) from the lysedcells. Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985, 79:277; Bruggemann et al., 1987, J Exp Med 166:1351; Wilkinson etal., 2001, J Immunol Methods 258:183; Patel et al., 1995, J InzinunoiMethods 184:29 and herein (see, e.g., section entitled “Characterizationand Functional Assays” infra). Alternatively, or additionally, ADCCactivity of the antibody may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., 1998, Proc. Nat'l Acad.Sci. USA 95:652.

Additionally, variant Rituximab antibodies or antibody fragments may bescreened for ADCP activity. For the phagocytosis experiments involved inthis assay, monocytes are isolated from PBMCs of individuals belongingto patient Group-II (FcγRIIIA V/F¹⁵⁸ and FcγRIIA H/H¹³¹) by using aPercoll gradient and differentiated into macrophages by culturing in amedium supplemented with 0.1 ng/ml granulocyte macrophagecolony-stimulating factor for 1 week. For imaging, WIL2-S target cellsare labeled with PKH67 (Sigma) and co-cultured for 24 hours withmacrophages at an effector:target cell ratio of 3:1 in the presence of100 ng/ml variant Rituximab. Cells are then treated with secondaryantibodies anti-CD11-RPE and anti-CD14-RPE (DAKO) for 15 minutes beforelive cell imaging using a fluorescence microscope. For quantitative ADCP(i.e., antibody-dependent cell-mediated phagocytosis; macrophagemediated ADCC), WIL2-S target cells are labeled with PKH67, seeded in a96-well plate at 20×10³ cells per well, and treated with WT or thevariant Rituximab at the designated final concentrations. Macrophagesare labeled with PKH26 (Sigma) and added to the opsonized labeled targetcells at 20×10³ cells per well, and the cells are co-cultured for 18 h.Fluorescence is measured by using dual-label flow cytometry. Variantantibodies or antibody fragments with increased potency and efficacy areselected. CDC assays are performed according to published procedures(Gazzano-Santoro et al., J. Immunol. Meth. 202:163, 1997; Uchida et al.,2004, J. Exp. Med. 199:1659).

Also, variant Rituximab antibodies or antibody fragments may be screenedfor those variant antibodies or antibody fragments that are capable ofdepleting B-cells in a subject. For instance, in vivo B-cell depletionassays may be employed. In such assays, Cynomolgus monkeys (Macacafascicularis) are injected intravenous once daily for 4 consecutive dayswith wild-type or the variant rituximab antibody (Reff et al., 1994,Blood 83:435-445). The test animals will be allotyped for the FcγRpolymorphisms, and if required, the animals corresponding to V/F¹⁵⁸ andH/H¹³¹ genotypes will be used for the experiment. The experiment iscomprised of six treatment Groups of .about.0.1, 0.2, 2, 7, or 30 μg/kg(variant Rituximab antibody) or .about.2 or 30 μg/kg (wild-typecontrol), with three monkeys per treatment Group. Blood samples areacquired on two separate days before dosing (baseline) and at days 1, 2,5, 15, and 28 after initiation of dosing. For each sample, cellpopulations are quantified by flow cytometry by using specificantibodies against the representative marker antigens. Percent B-celldepletion is calculated by comparing B-cell counts on the given day withthe average of the two baseline measures for each animal.

3. Selection of Variant Rituximab Antibodies Optimized for Group-II(V/F¹⁵⁸, H/H¹³¹ Genotype)

Variant Rituximab antibodies or antibody fragments are consideredoptimized for Group-II when they exhibit properties (e.g., bindingaffinity, ADCC activity, etc.) similar to those properties exhibited byGroups (genotypes) associated with a higher degree of responsiveness toRituxamab. In other embodiments, variant Rituximab antibodies orantibody fragments are considered optimized for Group-II when theyexhibit properties (i.e. binding affinity, ADCC activity, etc.) similarto those properties exhibited by the Group associated with the highestdegree of responsiveness to Rituximab (i.e. Group-1).

In some embodiments, variant antibodies or antibody fragments areconsidered optimized for Group-II when they exhibit a binding affinityto FcγRIIA and FcγRIIIA that is increased to a level exhibited by Groupswith a higher degree of responsiveness to Rituximab. In otherembodiments, variant antibodies or antibody fragments are consideredoptimized for Group-II when they exhibit a binding affinity to FcγRIIAand FcγRIIIA that is increased to the level exhibited by the Group withthe highest degree of responsiveness to Rituximab (Group-I).

In some embodiments, variant antibodies or antibody fragments areconsidered optimized for Group-II when they have ADCC activity similarto the ADCC activity exhibited by Groups with a higher degree ofresponsiveness to Rituximab. In other embodiments, variant antibodies orantibody fragments are considered optimized for Group-II when theyexhibit ADCC activity similar to the ADCC activity exhibited by theGroup with the highest degree of responsiveness to Rituximab (Group-I).

Variant Rituximab antibodies or antibody fragments that exhibit asimilar degree of responsiveness in the patients of Group-II as comparedto the responsiveness of Rituximab in the patients of Group-I areconsidered patient Group optimized. Variant antibodies optimized for aGroup-II are selected for use in the treatment of patients of Group-II.

Example 5 Enhancement of an Antibody's Effector Functions with aGenotype Optimized Fc

The Fc nucleotide sequence (Fc cassette) from an antibody optimized forresponsiveness for a particular FcγRIIA and FcγRIIIA genotype (e.g., bythe methods described in the above examples) can be used to optimize theresponsiveness of other antibodies used to treat the same or othersubjects with the particular FcγRIIA and FcγRIIIA genotype.

Fc cassettes with optimized responsiveness for each FcγRIIA (H/R¹³¹)polymorphism and FcγRIIIA (V/F¹⁵⁸) polymorphism are used to optimize theresponsiveness exhibited by other antibodies. As such, an Fc cassettethat is optimized for the V/F¹⁵⁸, H/H¹³¹ (Group-II) genotype can be usedto optimize the responsiveness of patients of the same genotype toanother antibody. For example, the optimized Fc cassette for the V/F¹⁵⁸,H/H¹³¹ (Group-II) genotype, engineered to optimize responsiveness toRituximab, is used to optimize the responsiveness in patients of thesame genotype to any other therapeutic antibody, such as HERCEPTIN®.

While the example described in detail below is directed towardoptimizing HERCEPTIN® for the V/F¹⁵⁸, H/H¹³¹ genotype, it will beunderstood that the same protocol may be employed to engineer optimizedantibodies for any other FcγRIIA and FcγRIIIA genotype.

Optimizing HERCEPTIN® for Group-II (V/F¹⁵⁸, H/H¹³¹)

HERCEPTIN® may be optimized for a patient with a Group-II genotype(V/F¹⁵⁸, H/H¹³¹) by fusion of the antigen binding domain of HERCEPTIN®to a Group-II optimized Fc cassette. Specifically, an Fc cassetteengineered to optimize responsiveness to an antibody (other than SHERCEPTIN®) for Group-II patients is used to optimize responsiveness toHERCEPTIN®.

As previously described, a variant Rituximab antibody is engineered foroptimized responsiveness in a patient with a Group-II genotype (V/F¹⁵⁸,H/H¹³¹). The optimized Fc portion of Rituximab is fused to the antigenbinding domain of HERCEPTIN® by recombinant techniques. Specifically,the Group-II optimized Rituximab Fc cassette is fused in frame with theantigen binding domain of HERCEPTIN® and inserted into an vector. Thevector may be transfected into an appropriate host cell and expressed toproduce the optimized Fc-HERCEPTIN®fusion. Any methods for generating anantibody fusion, such as, for example, those previously discussed, maybe employed.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of thedisclosure. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the disclosure andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the disclosure and theconcepts contributed by the inventors to furthering the art, and are tobe construed as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the disclosure as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentdisclosure, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent disclosure is embodied by the appended claims.

What is claimed is:
 1. A method for predicting treatment response to anantibody therapy for a human test subject having an ADCC treatabledisease or disorder, the method comprising: (a) classifying the testsubject into one of nine genotype groups for the combination of H/Rpolymorphisms at amino acid position 131 of FcγRIIA receptor; and F/Vpolymorphisms at amino acid position 158 of FcγRIIIA receptor; (b) usinga reference index that correlates more than three categories oftreatment response upon treatment with an antibody therapy with thegenotype groups; and (c) further comprising determining the ADCCfunction of the test subject.